Target-dependent transcription using deletion mutants of N4 RNA polymerase

ABSTRACT

The present invention comprises novel methods, compositions and kits that use N4 vRNAP deletion mutants to detect and quantify analytes comprising one or multiple target nucleic acid sequences, including target sequences that differ by as little as one nucleotide or non-nucleic acid analytes, by detecting a target sequence tag that is joined to an analyte-binding substance. The method consists of an annealing process, a DNA ligation process, an optional DNA polymerase extension process, a transcription process, and, optionally, a detection process

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a Continuation in Part of U.S. patentapplication Ser. No. 10/153,219, which claims priority to U.S.Provisional Patent Application Serial No. 60/292,845, filed May 22,2001. This application also claims priority to U.S. Provisional PatentApplication Serial No. 60/436,062 filed Dec. 23, 2002. The entiredisclosure of all priority applications is specifically incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] The government may own rights in the present invention pursuantto grant number R01 A1 12575 from the National Institute of Health.

BACKGROUND OF THE INVENTION

[0003] I. Field of the Invention

[0004] The present invention relates to novel methods, compositions andkits for amplifying, detecting and quantifying one or multiple targetnucleic acid sequences in a sample, including target sequences thatdiffer by as little as one nucleotide. The invention has broadapplicability for research, environmental and genetic screening, anddiagnostic applications, such as for detecting and quantifying sequencesthat indicate the presence of a pathogen, the presence of a gene or anallele, or the presence of a single nucleotide polymorphism (SNP) orother type of gene mutation or variant. The invention also relates tonovel methods, compositions and kits for detecting and quantifying abroad range of analytes by detecting a target sequence that is joined toan analyte-binding substance.

[0005] II. Description of Related Art

[0006] Transcription of DNA into mRNA is regulated by the promoterregion of the DNA. The promoter region contains a sequence of bases thatsignals RNA polymerase to associate with the DNA, and to initiate thetranscription of mRNA using one of the DNA strands as a template to makea corresponding complementary strand of RNA. RNA polymerases fromdifferent species typically recognize promoter regions comprised ofdifferent sequences. In order to obtain a transcription product by invitro or in vivo transcription, the promoter driving transcription ofthe gene or DNA sequence must be a cognate promoter for the RNApolymerase, meaning that it is recognized by the RNA polymerase.

[0007] There are a number of methods in the art for detecting nucleicacid sequences, including point mutations. The presence of a nucleicacid sequence can indicate, for example, the presence of a pathogen, orthe presence of particular genes or mutations in particular genes thatcorrelate with or that are indicative of the presence or status of adisease state, such as, but not limited to, a cancer.

[0008] Examples of methods that involve in vitro transcription formaking probes are described in: Murakawa et al., DNA 7:287-295, 1988;Phillips and Eberwine, Methods in Enzymol. Suppl. 10:283-288, 1996;Ginsberg et al., Ann. Neurol. 45:174-181, 1999; Ginsberg et al., Ann.Neurol. 48:77-87, 2000; VanGelder et al., Proc. Natl. Acad. Sci. USA87:1663-1667, 1990; Eberwine et al., Proc. Natl. Acad. Sci. USA89:3010-3014, 1992; U.S. Pat. Nos. 5,021,335; 5,168,038; 5,545,522;5,514,545; 5,716,785; 5,891,636; 5,958,688; 26,291,170; and PCT PatentApplications WO 00/75356 and WO 02/065093.

[0009] Still other methods use in vitro transcription as part of aprocess for amplifying and detecting one or more target nucleic acidsequences in order to detect the presence of a pathogen, such as a viralor microbial pathogen, that is a causative agent for a disease or todetect a gene sequence that is related to a disease or the status of adisease for medical purposes. Examples where in vitro transcriptionmethods have been used for medical purposes include U.S. Pat. Nos.5,130,238; 5,194,370; 5,399,491; 5,409,818; 5,437,990; 5,466,586;5,554,517; 5,665,545; 6,063,603; 6,090,591; 6,100,024; and 6,410,276;Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173, 1989; Fahy et al, In:PCR Methods and Applications, pp. 25-33, 1991; PCT Patent ApplicationNos. WO 89/06700 and WO 91/18155; and European Patent Application Nos.0427073 A2 and 0427074 A2.

[0010] Still other methods detect sequences or mutations using methodsthat involve ligation of adjacently hybridizing oligonucleotide probesor ligation of non-adjacently hybridizing probes following a processsuch as primer extension. Ligation detection methods include thosedisclosed in European Patent Application Publication Nos. 0246864 A2 and0246864 B1 of Carr; U.S. Pat. Nos. 4,883,750; 5,242,794; 5,521,065;5,962,223; and 6,054,266 of Whiteley, N. M. et al.; U.S. Pat. Nos.4,988,617 of Landegren and Hood; U.S. Pat. No. 5,871,921 of Landegrenand Kwiatkowski; U.S. Pat. No. 5,866,337 of Schon; European PatentApplication Publication Nos. 0320308 A2 and 0320308 B1 of Backman andWang; PCT Publication No. WO 89/09835 of Orgel and Watt and EuropeanPatent Publication No. 0336731 B1 of Bruce Wallace; U.S. Pat. No.5,686,272 of Marshall et al.; U.S. Pat. No. 5,869,252 of Bouma et al.;U.S. Pat. Nos. 5,494,810; 5,830,711; 6,054,564; 6,027,889; 6,268,148;and 6,312,892 of Barany et al.; U.S. Pat. Nos. 5,912,148 and 6,130,073of F. Eggerding; U.S. Pat. No. 6,245,505 B1 of Todd and Fuery; EuropeanPatent Application Publication No. 0357336 A2 of Ullman et al.; U.S.Pat. No. 5,427,930 of Birkenmeyer et al. and U.S. Pat. No. 5,792,607 andEuropean Patent Publication Nos. 0439182 A2 and EP 0439182 B1 of Backmanet al.; U.S. Pat. Nos. 5,679,524; and 5,952,174 of Nikifoorov et al.;U.S. Pat. No. 6,025,139 of Yager and Dunn; and U.S. Pat. No. 6,355,431B1 of Chee and Gunderson.

[0011] In addition, U.S. Pat. No. 6,153,384 of Lynch et al. discloses anassay to identify ligase activity modulators by ligation of a labelednucleic acid to an immobilized capture nucleic acid in the presence of apotential ligase activity modulator. Furthermore, Mahajan et al.,disclose in U.S. Pat. No. 5,976,806 a quantitative and functional DNAligase assay that uses a linearized plasmid containing a reporter gene,wherein ligase activity is followed by the extent of coupledtranscription-translation of the reporter gene.

[0012] Also, in U.S. Pat. No. 5,807,674 Tyagi discloses detection of RNAtarget sequences by ligation of the RNA binary probes, wherein asubstrate for Q-beta replicase is generated.

[0013] In PCT Patent Application No. WO 92/01813, Ruth and Driverdisclosed a process for synthesizing circular single-stranded nucleicacids by hybridizing a linear polynucleotide to a complementaryoligonucleotide and then ligating the linear polynucleotide. Theyfurther disclosed a process for generating multiple linear complementsof the circular single-stranded nucleic acid template by extending aprimer more than once around the circular template using a DNApolymerase.

[0014] Japanese Patent Nos. JP4304900 and JP4262799 of Toshiya et al.,disclose detection of a target sequence by ligation of a linearsingle-stranded probe having target-complementary 3′- and 5′-endsequences which are adjacent when the linear probe is annealed to atarget sequence in the sample, followed by either rolling circlereplication or in vitro transcription of the circular single-strandedtemplate. Toshiya et al., disclose that in vitro transcription isperformed by first annealing to the circular single-stranded template acomplementary nucleotide primer having an anti-promoter sequence inorder to form a double-stranded promoter, and then transcribing thecircular single-stranded template having the annealed anti-promoterprimer with an RNA polymerase that has helicase-like activity, such asT7, T3 or SP6 RNA polymerase.

[0015] In U.S. Pat. Nos. 6,344,329; 6,210,884; 6,183,960; 5,854,033;6,329,150; 6,143,495; 6,316,229; and 6,287,824, Paul M. Lizardi alsodisclose the use of rolling circle replication to amplify and detectnucleic acid sequences. Lizardi further describes use of RNA polymeraseprotopromoters in the circular probe so that tandem-sequencesingle-stranded protopromoter-containing DNA products resulting fromrolling circle replication can be transcribed by a cognate T7-type RNApolymerase following conversion of said DNA products to a formcontaining double-stranded promoters.

[0016] Furthermore, Kool et al., have disclosed synthesis of DNA or RNAmultimers, meaning multiple copies of an oligomer or oligonucleotidejoined end to end (i.e., in tandem) by rolling circle replication orrolling circle transcription, respectively, of a circular DNA templatemolecule. Rolling circle replication uses a primer and astrand-displacing DNA polymerase, such as phi 29 DNA polymerase. Withrespect to rolling circle transcription, it was shown these circularsingle-stranded DNA (ssDNA) molecules can be efficiently transcribed byphage and bacterial RNA polymerases (Prakash, G. and Kool, E., J. Am.Chem. Soc. 114: 3523-3527, 1992; Daubendiek, S. L. et al., J. Am. Chem.Soc. 117: 7818-7819, 1995; Liu, D. et al., J. Am. Chem. Soc. 118:1587-1594, 1996; Daubendiek, S. L. and Kool, E. T., Nature Biotechnol.,15: 273-277, 1997; Diegelman, A. M. and Kool, E. T., Nucleic Acids Res.,26: 3235-3241, 1998; Diegelman, A. M. and Kool, E. T., Chem. Biol., 6:569-576, 1999; Diegelman, A. M. et al., BioTechniques 25: 754-758, 1998;Frieden, M. et al., Angew. Chem. Int. Ed. Engl. 38: 3654-3657, 1999;Kool, E. T., Acc. Chem. Res., 31: 502-510, 1998; U.S. Pat. Nos.5,426,180; 5,674,683; 5,714,320; 5,683,874; 5,872,105; 6,077,668;6,096,880; and 6,368,802). Rolling circle transcription of thesecircular ssDNAs occurs in the absence of primers, in the absence of acanonical promoter sequence, and in the absence of any duplex DNAstructure, and results in synthesis of linear multimeric complementarycopies of the circle sequence up to thousands of nucleotides in length.Transcription of the linear precursor of the circular ssDNA templateyielded only a small amount of RNA transcript product that was shorterthan the template.

[0017] Fire and Xu (U.S. Pat. No. 5,648,245; Fire, A. and Xu, S-Q, Proc.Natl. Acad. Sci. USA, 92: 4641-4645, 1995) also disclose methods forusing rolling circle replication of small DNA circles to constructoligomer concatamers.

[0018] Other researchers, including, but not limited to, Mahtani (U.S.Pat. No. 6,221,603), Rothberg et al., (U.S. Pat. No. 6,274,320), Dean etal., (Genome Res., 11: 1095-1099, 2001), Lasken et al., (U.S. Pat. No.6,323,009), and Nilsson et al., (Nucleic Acids Res., 30 (14): e66, 2002)disclose other methods and applications of rolling circle amplification.Also, Pickering et al. (Nucleic Acids Res., 30 (12): e60, 2002)discloses a ligation and rolling circle amplification method forhomogeneous end-point detection of single nucleotide polymorphisms(SNPs).

[0019] Although a number of nucleic acid amplification methods have beendescribed in the art, there is a continuing need for methods and assaysfor detecting nucleic acids that are specific and accurate, yet areeasier and faster than current methods. The present invention providesnovel assays, methods, compositions and kits that are simple in formatand very rapid to perform, but that can be used to detect and quantifyany of a broad range of analytes with a high degree of specificity andsensitivity, including both nucleic acid analytes and non-nucleic acidanalytes. With respect to analytes comprising a target nucleic acid, theinvention provides assays, methods and kits that can detect anddistinguish between target sequences, including sequences that differeven by only a single nucleotide, such as for analysis of singlenucleotide polymorphisms.

[0020] All of the methods above for amplifying and detecting one or moretarget nucleic acid sequences use a double-stranded transcriptionpromoter. In contrast to the methods in the art, the present inventionprovides methods, compositions and kits for detecting target nucleicacid sequences using an RNA polymerase that uses single-stranded DNApromoters and templates and that lacks helicase-like activity, as wellas other advantages and benefits that will be clear from reading thespecification below.

BRIEF SUMMARY OF THE INVENTION

[0021] The invention provides methods of using a novel N4 virion RNApolymerase (vRNAP), a mini-vRNA polymerase, and compositions and relatedkits. The novel polymerases are described by an isolated nucleic acidcomprising a region encoding a polypeptide having the amino sequence setforth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ IDNO:15. The nucleic acid may comprise the nucleic acid sequence of SEQ IDNO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:14. Inpreferred embodiments of the invention, the RNA polymerase comprises atranscriptionally active 1,106-amino acid domain of the N4 vRNAP (hereindesignated “mini-vRNAP”), which corresponds to amino acids 998-2103 ofN4 vRNAP, as described herein. The vRNAP and mini-vRNA polymerasetranscribe nucleic acid operatively linked to an N4 promoter such as aP2 promoter of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:27, SEQ ID NO:28 orSEQ ID NO:29. The promoter of SEQ ID. NO:16 or SEQ ID NO:28 ispreferred.

[0022] The current invention can use a polypeptide encoded by anisolated polynucleotide comprising a sequence identical or complementaryto at least 14 contiguous nucleotides of SEQ ID NO:1. The polynucleotidemay comprise at least 20, 25, 30, 35, 40, 45, 50, 60, 75, 100, 150, 200,250, 300, 400, 600, 800, 1000, 2000, 3000, 3300 or more contiguousnucleotides of SEQ ID NO:1. The polynucleotide may comprise allcontiguous nucleotides of SEQ ID NO:3 or all contiguous nucleotides ofSEQ ID NO:1. Similarly, the polynucleotide may comprise at least 20, 25,30, 35, 40, 45, 50, 60, 75, 100, 150, 200, 250, 300, 400, 600, 800,1000, 2000, 3000, 3300 or more nucleotides complementary to at least 20,25, 30, 35, 40, 45, 50, 60, 75, 100, 150, 200, 250, 300, 400, 600, 800,1000, 2000, 3000, 3300 or more contiguous nucleotides of SEQ ID NO:1.

[0023] A purified N4 virion RNA polymerase of the current invention cancomprise at least 20 contiguous amino acids of SEQ ID NO:2. It ispreferred that the polymerase contain at least 25, 30, 35, 40, 45, 50,60, 75, 100, 150, 200, 250, 300, 400, 600, 800, 1000 or more contiguousamino acids of SEQ ID NO:2.

[0024] In another aspect, the current invention can use a polypeptideencoded by an isolated nucleic acid comprising a region encoding atleast 6 contiguous amino acids of SEQ ID NO:2, wherein the polypeptidehas RNA polymerase activity under appropriate reaction conditions. It ispreferred that this polypeptide comprises at least 10, 15, 20, 25, 30,35, 40, 45, 50, 60, 75, 100, 150, 200, 250, 300, 400, 600, 800, 1000 ormore contiguous amino acids of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, or SEQ ID NO:15. The encoded polypeptide may have at leastone hexahistidine tag or other tag, or the encoded polypeptide may lacka tag. The polypeptide may be a mutant of the peptide found in SEQ IDNO:2 or SEQ ID NO:4, such as an enzyme possessing an amino acidsubstitution at position Y678.

[0025] An embodiment of the current invention comprises a method ofmaking RNA. This method comprises: (a) obtaining a N4 virion RNApolymerase (i.e. the polypeptide); (b) obtaining DNA wherein the DNApreferably contains a N4 virion RNA polymerase promoter sequence; (c)admixing the RNA polymerase and the DNA; and (d) culturing the RNApolymerase and the DNA under conditions effective to allow RNAsynthesis. Optionally, the method may comprise synthesizingpolynucleotides containing modified ribonucleotides ordeoxyribonucleotides. The DNA is preferably single-stranded DNA ordenatured double-stranded DNA. Step (c) may occur in a host cell such asan E. coli host cell.

[0026] The amino acid sequence of the RNA polymerase is preferably thesequence essentially as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:15, or a mutant form of the polymerase ofSEQ ID NO:4 or SEQ ID NO:6. The mutation may be, for example, atposition number Y678. The RNA transcript may contain derivatizednucleotides.

[0027] An aspect of the current invention comprises using an N4 vRNAPpromoter to direct transcription. The promoter is preferentially an N4promoter set forth in SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:27, SEQ ID NO:28 or SEQ ID NO:29. The P2 promoter of SEQ ID NO:16 orSEQ ID NO:28 is preferred. The promoter sequence may be upstream of thetranscription initiation site. The promoter may comprise a set ofinverted repeats forming a hairpin with a 2-7 base pair long stem and3-5 base loop having purines in the central and/or next to the centralposition of the loop.

[0028] The preferred conditions of the transcription method claimedherein includes a pH in step (c) of between 6 and 9, with a pH ofbetween 7.5 and 8.5 more preferred. Mg⁺² or Mn⁺², preferably Mg⁺² may beadmixed. Preferred temperatures for the reaction are 25° C. to 50° C.with the range of 30° C. to 45° C. being more preferred and the range of32° C. to 42° C. being most preferred. The admixing may occur in vivo orin vitro.

[0029] An aspect of the current invention also includes translation ofthe RNA after transcription. A reporter gene such as an α-peptide ofβ-galactosidase may be used. It is preferred the transcription comprisesadmixing an E. coli single-stranded binding protein (EcoSSB), a SSBprotein homologous to EcoSSB or another naturally occurring or chimericSSB protein homologous to EcoSSB with the polymerase and DNA. Yetanother aspect of the current invention is the transcription method inwhich no EcoSSB is admixed with the RNA polymerase and DNA; the productof this method is a DNA/RNA hybrid.

[0030] The DNA admixed with the RNA polymerase of the current inventionmay be single-stranded linear DNA or single-stranded circular DNA suchas bacteriophage M13 DNA. The DNA may be denatured DNA, such assingle-stranded, double-stranded linear or double-stranded circulardenatured DNA. The DNA may also be double-stranded DNA under certainconditions. The RNA may be pure RNA or may contain modified nucleotides.Mixed RNA-DNA oligonucleotides may also be synthesized with the Y678Fmutant mini-vRNAP (SEQ ID NO:8) of the current invention.

[0031] The synthesized RNA may comprise a detectable label such as afluorescent tag, biotin, digoxigenin, 2′-fluoro nucleoside triphosphate,or a radiolabel such as a ³⁵S- or ³²P-label. The synthesized RNA may beadapted for use as a probe for blotting experiments or in-situhybridization. Nucleoside triphosphates (NTPs) or derivatized NTPs maybe incorporated into the RNA, and may optionally have a detectablelabel. Deoxynucleoside triphosphates may be incorporated into the RNA.

[0032] The RNA may be adapted for use in NMR structural determination.Short RNAs such as those between 10 and 1000 bases or between 10 and 300bases may be used. The RNA may be adapted for use in spliceosomeassembly, splicing reactions or for antisense or RNA interferenceexperiments. Also, the RNA may be adapted for use in probing for acomplementary nucleotide sequence or for use as a probe in RNaseprotection studies.

[0033] Yet another aspect of the current invention comprises deliveringRNA into a cell after transcription of the RNA. The delivery may be bymicroinjection, transfection, electroporation, or other methods in theart. Another aspect of the invention comprises amplifying the RNA aftertranscription.

[0034] Another embodiment of the current invention comprises a method ofmaking RNA comprising: (a) obtaining a N4 virion RNA polymerase; (b)obtaining a single-stranded DNA oligonucleotide wherein theoligonucleotide contains a N4 virion RNA polymerase promoter sequence;(c) admixing the RNA polymerase and the oligonucleotide; and (d)culturing the RNA polymerase and the oligonucleotide under conditionseffective to allow RNA synthesis. The polymerase preferentially has theamino sequence set forth in SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8. Inthis embodiment, it is preferred that the DNA has between 20 and 200bases.

[0035] Yet another embodiment of the invention comprises a method ofmaking RNA comprising: (a) obtaining a N4 virion RNA polymerase; (b)obtaining a single-stranded DNA wherein the DNA contains a N4 virion RNApolymerase promoter sequence; (c) obtaining a ribonucleosidetriphosphate (XTP) or a derivatized ribonucleoside triphosphate; (d)admixing the RNA polymerase, the DNA and the XTP; and (e) culturing theRNA polymerase and the oligonucleotide under conditions effective toallow RNA synthesis wherein the RNA is a derivatized RNA. The RNApolymerase preferentially has the amino sequence set forth in SEQ IDNO:4 or SEQ ID NO:6 or a mutant of the polymerase of SEQ ID NO:4 or SEQID NO:6, such as a mutant with a mutation at position number Y678 or thepolymerase of SEQ ID NO:8.

[0036] One embodiment comprises a method to detect a target nucleic acidsequence, the method comprising a DNA ligation operation and atranscription operation, wherein the DNA ligation operation comprisesligation of one or more target probes comprising a promoter that thatbinds an RNA polymerase that can bind a single-stranded promoter andinitiate transcription therefrom, wherein the ligation is dependent onhybridization of the target probes to the target nucleic acid sequence,and wherein the transcription operation comprises contacting thetranscription substrate with an RNA polymerase that binds thesingle-stranded promoter under transcription condition to obtain atranscription product. In preferred embodiments of this aspect of theinvention, the RNA polymerase comprises a transcriptionally active1,106-amino acid domain corresponding to amino acids 998-2103 of N4vRNAP. Preferably, the enzyme lacks a histidine or other tag. An RNApolymerase in various embodiments of the invention can also have theamino sequence set forth in SEQ ID NO:4 or SEQ ID NO:6 or a mutant ofthe polymerase of SEQ ID NO:4 or SEQ ID NO:6, such as a mutant with amutation at position number Y678 or the polymerase of SEQ ID NO:8, or itcan have a transcriptionally active portion of any of these sequences.In some embodiments, the target probes comprise monopartite targetprobes comprising a promoter target probe and a signal target probeand/or optionally, one or more simple target probes. In otherembodiments, a bipartite target probe and, optionally, one or moresimple target probes is used. In some embodiments, the target sequencecomprises a target nucleic acid in a sample, whereas in otherembodiments the target sequence comprises a target sequence tag that isjoined to an analyte-binding substance that binds an analyte in thesample. In some embodiments in which a bipartite target probe is used,the transcription substrate that is transcribed remains catenated to atarget nucleic acid. In other embodiments of methods in which abipartite target probe is used, the target sequence is preferably lessthan about 150 to about 200 nucleotides from the 3′-end of the targetnucleic acid or target sequence tag. In still other embodiments ofmethods in which a bipartite target probe is used and in which thetarget sequence is greater than about 150 to about 200 nucleotides fromthe 3′-end of the target nucleic acid or target sequence tag comprisingthe target sequence, one or more additional steps is used in order torelease the catenated circular ligation product from the target sequenceprior to transcription, as described elsewhere herein.

[0037] One aspect of this embodiment of the invention comprises a methodfor detecting a target nucleic acid sequence, the method comprising: (a)providing one or more target probes comprising linear single-strandedDNA, the target probes comprising at least two target-complementarysequences that are not joined to each other, wherein the 5′-end of afirst target-complementary sequence is complementary to the 5′-end ofthe target nucleic acid sequence, and wherein the 3′-end of a secondtarget-complementary sequence is complementary to the 3′-end of thetarget nucleic acid sequence, and wherein the target probe thatcomprises the first target-complementary sequence also comprises apromoter that is joined to the 3′-end of the first target complementarysequence, which promoter can bind a single-stranded promoter andinitiate transcription therefrom; (b) contacting the target probes withthe target nucleic acid sequence and incubating under hybridizationconditions, wherein the target-complementary sequences anneal adjacentlyto the target nucleic acid sequence to form a complex; (c) contactingthe complex with a ligase under ligation conditions to form atranscription substrate; (d) contacting the transcription substrate withan RNA polymerase that can bind the single-stranded promoter undertranscription conditions to obtain a transcription product; and (e)detecting the transcription product. In preferred embodiments of thisaspect of the invention, the RNA polymerase comprises atranscriptionally active 1,106-amino acid domain corresponding to aminoacids 998-2103 of N4 vRNAP. Preferably, the enzyme lacks a histidine orother tag. An RNA polymerase in various embodiments of the invention canalso have the amino sequence set forth in SEQ ID NO:4 or SEQ ID NO:6 ora mutant of the polymerase of SEQ ID NO:4 or SEQ ID NO:6, such as amutant with a mutation at position number Y678 or the polymerase of SEQID NO:8, or it can have a transcriptionally active portion of any ofthese sequences. In some embodiments, the target probes comprisemonopartite target probes comprising a promoter target probe and asignal target probe and/or optionally, one or more simple target probes.In other embodiments, a bipartite target probe and, optionally, one ormore simple target probes is used. In some embodiments, the targetsequence comprises a target nucleic acid in a sample, whereas in otherembodiments the target sequence comprises a target sequence tag that isjoined to an analyte-binding substance that binds an analyte in thesample. In some embodiments in which a bipartite target probe is used,the transcription substrate that is transcribed remains catenated to atarget nucleic acid. In other embodiments of methods in which abipartite target probe is used, the target sequence is preferably lessthan about 150 to about 200 nucleotides from the 3′-end of the targetnucleic acid or target sequence tag. In still other embodiments ofmethods in which a bipartite target probe is used and in which thetarget sequence is greater than about 150 to about 200 nucleotides fromthe 3′-end of the target nucleic acid or target sequence tag comprisingthe target sequence, one or more additional steps is used in order torelease the catenated circular ligation product from the target sequenceprior to transcription, as described elsewhere herein.

[0038] Another aspect of this embodiment of the invention comprises amethod for detecting a target nucleic acid sequence, the methodcomprising: (a) providing one or more target probes comprising linearsingle-stranded DNA, the target probes comprising at least twotarget-complementary sequences that are not joined to each other,wherein the 5′-end of a first target-complementary sequence iscomplementary to the 5′-end of the target nucleic acid sequence, andwherein the 3′-end of a second target-complementary sequence iscomplementary to the 3′-end of the target nucleic acid sequence, andwherein the target probe that comprises the first target-complementarysequence also comprises a promoter that is joined to the 3′-end of thefirst target-complementary sequence, which promoter binds an RNApolymerase that can bind a single-stranded promoter and initiatetranscription therefrom; (b) contacting the target probes with thetarget nucleic acid sequence and incubating under hybridizationconditions whereby the target probes anneal to the target nucleic acidsequence to form a complex; (c) contacting the complex with a DNApolymerase under DNA polymerization conditions to form a DNA polymeraseextension product that is contiguous with the 5′-end of the firsttarget-complementary sequence; (d) contacting the DNA polymeraseextension product complex with a ligase under ligation conditions toform a transcription substrate; (e) contacting the transcriptionsubstrate with an RNA polymerase that can bind a single-strandedpromoter and initiate transcription therefrom under transcriptioncondition to obtain a transcription product; and (f) detecting thetranscription product. In preferred embodiments of this aspect of theinvention, the RNA polymerase comprises a transcriptionally active1,106-amino acid domain corresponding to amino acids 998-2103 of N4vRNAP. Preferably, the enzyme lacks a histidine or other tag. An RNApolymerase in various embodiments of the invention can also have theamino sequence set forth in SEQ ID NO:4 or SEQ ID NO:6 or a mutant ofthe polymerase of SEQ ID NO:4 or SEQ ID NO:6, such as a mutant with amutation at position number Y678 or the polymerase of SEQ ID NO:8, or itcan have a transcriptionally active portion of any of these sequences.In some embodiments, the target probes comprise monopartite targetprobes comprising a promoter target probe and a signal target probeand/or optionally, one or more simple target probes. In otherembodiments, a bipartite target probe and, optionally, one or moresimple target probes is used. In embodiments of methods in which abipartite target probe is used, the target sequence is preferably lessthan about 150 to about 200 nucleotides from the 3′-end of the targetnucleic acid or target sequence tag. In some embodiments of methods inwhich a bipartite target probe is used and in which the target sequenceis greater than about 150 to about 200 nucleotides from the 3′-end ofthe target nucleic acid or target sequence tag comprising the targetsequence, one or more additional steps is used in order to release thecatenated circular ligation product from the target sequence prior totranscription, as described elsewhere herein. In other embodiments inwhich a bipartite target probe is used, the transcription substrate thatis transcribed remains catenated to a target nucleic acid. In someembodiments, the target sequence comprises a target nucleic acid in asample, whereas in other embodiments the target sequence comprises atarget sequence tag that is joined to an analyte-binding substance thatbinds an analyte in the sample.

[0039] Another embodiment of the invention comprises a method forobtaining transcription products comprising multiple copies of a targetnucleic acid sequence (target sequence) in a sample, said methodcomprising: (a) providing one or more target probes comprising linearsingle-stranded DNA, said one or more target probes having at least twodifferent target-complementary sequences that are not joined to eachother, wherein the 5′-end of a first target-complementary sequence iscomplementary to the 5′-end of the target sequence and the 3′-end of asecond target-complementary sequence is complementary to the 3′-end ofthe target sequence, and wherein the target probe that comprises atarget-complementary sequence that is complementary to the 5′-end of thetarget sequence also comprises a promoter that is 3′- of thetarget-complementary sequence of said target probe, which promoter isfor an RNA polymerase that lacks helicase-like activity and that canbind said single-stranded promoter and initiate transcription therefromunder transcription conditions, and wherein any additional targetprobes, if provided, comprise simple target probes havingtarget-complementary sequences that anneal to the target sequencebetween the annealing sites of the first target-complementary sequenceand the second target-complementary sequence, and wherein every free5′-end of a target-complementary sequence that anneals to a targetsequence has a 5′-phosphate and is adjacent to a 3′-end of anothertarget-complementary sequence that has a 3′-hydroxyl end; (b) contactingthe target probes with the target sequence and incubating underhybridization conditions so as to permit the target-complementarysequences of said target probes to anneal adjacently to all portions ofthe target sequence; (c) contacting said target probes annealed to saidtarget sequence with a ligase under ligation conditions, wherein saidligase has little or no activity in ligating blunt ends and issubstantially more active in ligating ends that are adjacent whenannealed to two contiguous regions of a target sequence compared to endsthat are not annealed to said target sequence, so as to obtain a ligatedsingle-stranded DNA polynucleotide that comprises a transcriptionsubstrate for an RNA polymerase that lacks helicase-like activity andthat can bind the single-stranded promoter in said transcriptionsubstrate and initiate transcription therefrom under transcriptionconditions; and (d) obtaining said transcription substrate, wherein saidtranscription substrate comprises a sequence that is complementary tosaid target sequence; and (e) contacting said transcription substratewith the RNA polymerase that can bind said promoter and initiatetranscription therefrom under transcription conditions so as to obtaintranscription product that is complementary to said transcriptionsubstrate; and (f) detecting synthesis of said transcription productcomprising multiple copies of the target sequence obtained fromtranscription of said transcription substrate under transcriptionconditions, wherein synthesis of said transcription product indicatesthe presence of the target sequence. In preferred embodiments of thisaspect of the invention, the RNA polymerase comprises atranscriptionally active 1,106-amino acid domain corresponding to aminoacids 998-2103 of N4 vRNAP. Preferably, the enzyme lacks a histidine orother tag. An RNA polymerase in various embodiments of the invention canalso have the amino sequence set forth in SEQ ID NO:4 or SEQ ID NO:6 ora mutant of the polymerase of SEQ ID NO:4 or SEQ ID NO:6, such as amutant with a mutation at position number Y678 or the polymerase of SEQID NO:8, or it can have a transcriptionally active portion of any ofthese sequences. In some embodiments, the target probes comprisemonopartite target probes comprising a promoter target probe and asignal target probe and/or optionally, one or more simple target probes.In other embodiments, a bipartite target probe and, optionally, one ormore simple target probes is used. In embodiments of methods in which abipartite target probe is used, the target sequence is preferably lessthan about 150 to about 200 nucleotides from the 3′-end of the targetnucleic acid or target sequence tag. In some embodiments of methods inwhich a bipartite target probe is used and in which the target sequenceis greater than about 150 to about 200 nucleotides from the 3′-end ofthe target nucleic acid or target sequence tag comprising the targetsequence, one or more additional steps is used in order to release thecatenated circular ligation product from the target sequence prior totranscription, as described elsewhere herein. In other embodiments inwhich a bipartite target probe is used, the transcription substrate thatis transcribed remains catenated to a target nucleic acid. In someembodiments, the target sequence comprises a target nucleic acid in asample, whereas in other embodiments the target sequence comprises atarget sequence tag that is joined to an analyte-binding substance thatbinds an analyte in the sample.

[0040] Another embodiment of the present invention comprises a methodfor obtaining a transcription product complementary to a target nucleicacid sequence (target sequence), said method comprising: (a) providing atarget sequence amplification probe (TSA probe), wherein said TSA probecomprises a linear single-stranded DNA (ssDNA) comprising two endportions that are complementary to a contiguous target sequence andwhich end portions are connected by an intervening sequence, and whereinsaid TSA probe can form a TSA circle upon joining of said ends; (b)providing a primer that is complementary to the intervening sequence ofsaid TSA probe; (c) providing a bipartite target probe, wherein saidbipartite target probe comprises a linear ssDNA comprising two endportions that are complementary to a contiguous target sequence, andwherein said bipartite target probe forms a circular transcriptionsubstrate upon joining of said ends; (d) annealing said TSA probe tosaid target sequence under hybridization conditions; (e) ligating saidTSA probe annealed to said target sequence with a ligase under ligationconditions, wherein said ligase has little or no activity in ligatingblunt ends and is substantially more active in ligating said ends ofsaid bipartite target probe if said ends are adjacent when annealed totwo contiguous regions of a target sequence than if said ends are notannealed to said target sequence, so as to obtain a TSA circle; (f)annealing the primer that is complementary to the intervening sequenceof the TSA probe to the TSA circle under hybridization conditions; (g)contacting said TSA circle to which said primer is annealed with astrand-displacing DNA polymerase under strand-displacing polymerizationconditions so as to obtain a rolling circle replication productcomprising multiple copies of the target sequence; (h) annealing saidbipartite target probe to said multiple copies of the target sequence ofsaid rolling circle replication product under hybridization conditions;(i) ligating said bipartite target probe annealed to said multiplecopies of the target sequence of said rolling circle replication productwith a ligase under ligation conditions, wherein said ligase has littleor no activity in ligating blunt ends and is substantially more activein ligating ends that are adjacent when annealed to two contiguousregions of a target sequence than if said ends are not annealed, so asto obtain a circular ssDNA molecule that comprises a circulartranscription substrate; (j) obtaining said circular transcriptionsubstrate, wherein said circular transcription substrate comprises asequence that is complementary to said target sequence; (k) contactingsaid circular transcription substrate with an RNA polymerase undertranscription conditions so as to obtain a transcription product that iscomplementary to said circular transcription substrate; (l) obtainingsaid transcription product that is complementary to said circulartranscription substrate, wherein said transcription product indicatesthe presence of said target sequence. In preferred embodiments of thisaspect of the invention, the RNA polymerase comprises atranscriptionally active 1,106-amino acid domain corresponding to aminoacids 998-2103 of N4 vRNAP. Preferably, the enzyme lacks a histidine orother tag. An RNA polymerase in various embodiments of the invention canalso have the amino sequence set forth in SEQ ID NO:4 or SEQ ID NO:6 ora mutant of the polymerase of SEQ ID NO:4 or SEQ ID NO:6, such as amutant with a mutation at position number Y678 or the polymerase of SEQID NO:8, or it can have a transcriptionally active portion of any ofthese sequences. In some embodiments, the target sequence comprises atarget nucleic acid in a sample, whereas in other embodiments the targetsequence comprises a target sequence tag that is joined to ananalyte-binding substance that binds an analyte in the sample. Inembodiments of methods in which a TSA probe or a bipartite target probeis used, the target sequence is preferably less than about 150 to about200 nucleotides from the 3′-end of the target nucleic acid or targetsequence tag. In some embodiments, the TSA circle that is replicatedremains catenated to the target nucleic acid or target sequence tag. Inother embodiments of methods in which the target sequence is greaterthan about 150 to about 200 nucleotides from the 3′-end of the targetnucleic acid or target sequence tag, then one or more additional stepsis used in order to release the catenated TSA circles from the targetsequence prior to rolling circle replication, as described elsewhereherein. Similarly, one or more additional steps can be used in order torelease the catenated circular ssDNA ligation products that result fromligation of bipartite target probes that are annealed to targetsequences in the rolling circle replication product more than about 100nucleotides to about 150 nucleotides from the 3′-end of to the rollingcircle replication product.

[0041] Yet another embodiment is a method for detecting a targetsequence, said method comprising: (a) providing a first bipartite targetprobe, wherein said first bipartite target probe comprises a 5′-portionand a 3′-portion, wherein said 5′-portion comprises: (i) a 5′-endportion that comprises a sequence that is complementary to a targetsequence, and (ii) a promoter sequence, wherein said promoter sequenceis covalently attached to and 3′- of said target-complementary sequencein said 5′-portion; and wherein said 3′-portion comprises: (i) a 3′-endportion that comprises a sequence that is complementary to a targetsequence, wherein said target-complementary sequence of said 3′-endportion, when annealed to said target sequence, is adjacent to saidtarget-complementary sequence of said 5′-end portion of said firstbipartite target probe, and (ii) optionally, a signal sequence, whereinsaid signal sequence is 5′- of said target-complementary sequence ofsaid 3′-portion of said first bipartite target probe; (b) providing asecond bipartite target probe, wherein said second bipartite targetprobe comprises a 5′-portion and a 3′-portion, wherein said 5′-portioncomprises: (i) a 5′-end portion that comprises sequence that iscomplementary to said target-complementary sequence of said 3′-endportion of said first bipartite target probe, and (ii) a promotersequence, wherein said promoter sequence in said 5′-portion of saidsecond bipartite target probe is 3′- of said target-complementarysequence in said 5′-portion; and wherein said 3′-portion comprises: (i)a 3′-end portion that comprises sequence that is complementary to saidtarget-complementary sequence of said 5′-end portion of said firstbipartite target probe, and (ii) optionally, a signal sequence, whereinsaid signal sequence in said 3′-portion of said second bipartite targetprobe is 5′- of said target-complementary sequence in said 3′-portion;(c) annealing said first bipartite target probe to the target sequenceunder hybridization conditions; (d) ligating said first bipartite targetprobe annealed to said target sequence with a ligase under ligationconditions, wherein said ligase has little or no activity in ligatingblunt ends and is substantially more active in ligating said ends ofsaid first bipartite target probe if said ends are adjacent whenannealed to two contiguous regions of a target sequence than if saidends are not annealed to said target sequence, so as to obtain acircular ssDNA molecule that comprises a first circular transcriptionsubstrate; (e) obtaining said first circular transcription substrate;(f) contacting said first circular transcription substrate with an RNApolymerase under transcription conditions so as to synthesizetranscription product that is complementary to said first circulartranscription substrate; (g) annealing to said transcription productthat is complementary to said first circular transcription substrate aprimer, wherein said primer is complementary to said transcriptionproduct; (h) contacting said transcription product to which said primeris annealed with a reverse transcriptase under reverse transcriptionconditions so as to obtain a first first-strand cDNA; (i) obtaining saidfirst first-strand cDNA, wherein said first first-strand cDNA comprisesa linear transcription substrate; (j) annealing to said firstfirst-strand cDNA said second bipartite target probe under annealingconditions; (k) contacting said first first-strand cDNA to which saidsecond bipartite target probe is annealed with a ligase under ligationconditions, wherein said ligase has little or no activity in ligatingblunt ends and is substantially more active in ligating said ends ofsaid second bipartite target probe if said ends are adjacent whenannealed to two contiguous regions of said first first-strand cDNA thanif said ends are not annealed to said sequence, so as to obtain acircular ssDNA molecule that comprises a second circular transcriptionsubstrate; (l) obtaining said second circular transcription substrate;(m) contacting said second circular transcription substrate with an RNApolymerase under transcription conditions so as to synthesizetranscription product that is complementary to said second circulartranscription substrate; (n) annealing to said transcription productthat is complementary to said second circular transcription substrate aprimer, wherein said primer is complementary to said transcriptionproduct; (o) contacting said transcription product to which said primeris annealed with a reverse transcriptase under reverse transcriptionconditions so as to obtain a second first-strand cDNA; (p) obtainingsaid second first-strand cDNA, wherein said second first-strand cDNAcomprises a linear transcription substrate; (q) annealing to said secondfirst-strand cDNA said first bipartite target probe under annealingconditions; (r) contacting said second first-strand cDNA to which saidfirst bipartite target probe is annealed with a a ligase under ligationconditions, wherein said ligase has little or no activity in ligatingblunt ends and is substantially more active in ligating said ends ofsaid first bipartite target probe if said ends are adjacent whenannealed to two contiguous regions of said second first-strand cDNA thanif said ends are not annealed to said sequence, so as to obtain acircular ssDNA molecule that comprises a third circular transcriptionsubstrate that is identical to said first circular transcriptionsubstrate; (s) obtaining said third circular transcription substratethat is identical to said first circular transcription substrate; (t)repeating steps (a) through (t); (u) detecting the synthesis oftranscription products resulting from transcription of said first,second and third circular transcription substrates and from said firstand second linear transcription substrates, wherein said synthesis ofsaid transcription products indicates the presence of said targetsequence comprising said target nucleic acid. In preferred embodimentsof this aspect of the invention, the RNA polymerase comprises atranscriptionally active 1,106-amino acid domain corresponding to aminoacids 998-2103 of N4 vRNAP. Preferably, the enzyme lacks a histidine orother tag. An RNA polymerase in various embodiments of the invention canalso have the amino sequence set forth in SEQ ID NO:4 or SEQ ID NO:6 ora mutant of the polymerase of SEQ ID NO:4 or SEQ ID NO:6, such as amutant with a mutation at position number Y678 or the polymerase of SEQID NO:8, or it can have a transcriptionally active portion of any ofthese sequences. In some embodiments, the target sequence comprises atarget nucleic acid in a sample, whereas in other embodiments the targetsequence comprises a target sequence tag that is joined to ananalyte-binding substance that binds an analyte in the sample.

[0042] Another embodiment of the invention comprises a method for invivo or in vitro protein synthesis comprising: (a) obtaining an RNApolymerase having the amino sequence set forth in SEQ ID NO:4, SEQ IDNO:6 or a mutant thereof; (b) obtaining DNA wherein the DNA contains aN4 virion RNA polymerase promoter sequence; (c) admixing the RNApolymerase and the DNA; (d) culturing the RNA polymerase and the DNAunder conditions effective to allow RNA synthesis; and (e) culturing theRNA in vivo or in vitro under conditions effective to allow proteinsynthesis. Step (e) may comprise using a two plasmid system or a oneplasmid system in which a reporter gene and the RNA polymerase gene arelocated on the same plasmid.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0043] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0044]FIG. 1—Bacteriophage N4 vRNAP promoters on single-strandedtemplates. These promoters are characterized by conserved sequences anda 5 bp stem, 3 base loop hairpin structure.

[0045]FIG. 2A and FIG. 2B—N4 vRNAP and generation of mini-vRNAP. FIG. 2Ashows a schematic of the N4 vRNAP protein with three motifs: the T/DxxGRmotif found in DNA-dependent polymerases, the P-loop, an ATP/GTP-bindingmotif present in some nucleotide-binding proteins, and motif B(Rx₃Kx₆₋₇YG), one of three motifs common to the Pol I and Pol α DNApolymerases and the T7-like RNA polymerases. FIG. 2B shows themini-vRNAP.

[0046]FIG. 3A and FIG. 3B—Identification of the minimaltranscriptionally active domain of N4 vRNAP by proteolytic cleavage.FIG. 3A, SDS-PAGE analysis of the products of vRNAP digestion withtrypsin. FIG. 3B N-terminal sequencing of the three initial proteolyticfragments indicated that the stable active polypeptide (mini-vRNAP)corresponds to the middle ⅓ of vRNAP, the region containing the threemotifs described in FIG. 2A.

[0047]FIG. 4—ORFs for full length polymerase, mini-vRNAP and mutantsthereof were cloned under pBAD control with an N-terminal hexahistidinetag. In other experiments, ORFs for mini-vRNAP and the Y678F mutantthereof, both of which lacked an N-terminal hexahistidine tag, werecloned in E. coli under pT7 control in cells which inducibly express T7RNAP; the polypeptides obtained showed similar activities to thepolypeptides with hexahistidine tags shown in this figure.

[0048]FIG. 5—Purification of cloned vRNAP and mini-vRNAP. The left handside shows the relative amounts of full size and mini-vRNAP proteinspurified on TALON columns from the same volume of induced cells. Furtherconcentration on a monoQ column reveals that, in contrast to full sizevRNAP, mini-vRNAP is stable after induction (right).

[0049]FIG. 6—Activation of N4 vRNAP transcription by EcoSSB at threedifferent ssDNA concentrations. The extent of EcoSSB activation istemplate-concentration dependent, with highest activation at low DNAtemplate concentration.

[0050]FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D—Effect of EcoSSB on ssDNAtemplate recycling. In the absence of EcoSSB, no increase intranscription was observed beyond 10 min of incubation (FIG. 7A).Addition of template at 20 min to the reaction carried out in theabsence of EcoSSB led to a dramatic increase in RNA synthesis (FIG. 7B).RNA synthesis increased linearly throughout the period of incubation(FIG. 7C). Addition of EcoSSB at 20 min led to a slow rate oftranscriptional recovery (FIG. 7D).

[0051]FIG. 8—Effect of EcoSSB on the state of template DNA and productRNA in vRNAP transcription. Native gel electrophoresis was carried outin the absence and in the presence of EcoSSB. Transcription wasperformed at an intermediate (5 nM) DNA concentration, at which only a2-fold effect of EcoSSB is observed. Either 32P-labeled template (rightpanel) or labeled NTPs (left panel) were used to analyze the state ofthe template (right panel) or RNA product (left panel) in the absence orpresence of EcoSSB.

[0052]FIG. 9A, FIG. 9B, and FIG. 9C—Transcription initiation by vRNAPand mini-vRNAP. The initiation properties of the full length andmini-vRNA polymerases were compared at similar molar concentrations(FIG. 9A) using the catalytic autolabeling assay and two reactionconditions: using a template containing +IC, the benzaldehyde derivativeof GTP and α-³²P-ATP, or a template containing +1T, the benzaldehydederivative of ATP and α-³²P-GTP. Comparison of the results in FIGS. 9Band 9C demonstrates that mini-vRNAP exhibits initiation propertiessimilar to full size vRNAP.

[0053]FIG. 10—Effect of EcoSSB on transcription of vRNAP and mini-vRNAP.The elongation and termination properties of vRNAP and mini-vRNAP arecompared.

[0054]FIG. 11A and FIG. 11B—Determination of mini-vRNAP promotercontacts. A 20-base oligonucleotide containing wild type promoter P2sequence binds with a 1 nM Kd (FIG. 11A). Most oligonucleotidessubstituted with 5-Iodo-dU at specific positions showed close to wildtype affinity except for the oligonucleotides substituted at positions−11 (at the center of the loop) and −8, indicating that these positionsare essential for promoter recognition (FIG. 11B). UV crosslinkingindicates that mini-vRNAP primarily contacts the −11 position.

[0055]FIG. 12—Binding affinities of stem-length promoter mutants. Wildtype promoter P2 with a 5 bp stem has a Kd of 1 nM (top). The stem wasshortened by removal of 3′ bases (left). The stem can be shortened bytwo base pairs without change in the binding affinity. The effect oflengthening the stem by addition of 3′ bases is shown (right). The stemcan be lengthened by two base pairs without change in the bindingaffinity.

[0056]FIG. 13A and FIG. 13B—Identification of the transcription startsite by catalytic autolabeling. A series of templates were constructedwith a single C placed at different distances from the center of thehairpin (position −11) by addition or deletion of the tract of Aspresent at promoter P2 (FIG. 13A). The affinity of mini-vRNAP for thesepromoters was measured by filter binding, and transcription initiationwas measured by catalytic autolabeling of mini-vRNAP. All templatesshowed similar binding affinities. However, only the template with a Cpositioned 12 bases downstream from the center of the hairpin was ableto support transcription initiation (FIG. 13B).

[0057]FIG. 14—UV crosslinking of mutant mini-vRNAPases to promoteroligonucleotides. Two mutants (K670A and Y678F) were tested for theirability to bind to wild type promoters. Both mutant RNA polymerasesbound to promoter DNA with wild type affinities and crosslinked to5-Iodo-dU substituted P2 DNA templates at positions −11 and +3 as wellas the wild type enzyme, indicating that these polymerase mutations donot affect promoter binding.

[0058]FIG. 15—Run-off transcription by mutant mini-vRNAPases. The wildtype and Y678F (SEQ ID NO:8) enzymes displayed similar activities atboth template excess and template-limiting conditions, while the K670Aenzyme exhibited decreased activity under both conditions. Underlimiting template conditions, all three enzymes were activated by EcoSSB(right panel). However, the Y678F enzyme showed reduced discriminationbetween incorporation of ribo- and deoxyribonucleoside triphosphates.

[0059]FIG. 16—Mutant mini-vRNAPases in transcription initiation. Theinitiation properties of the three enzymes were compared using catalyticautolabeling. The K670A enzyme displays significantly reduced activitywith the GTP derivative. The Y678F enzyme, in contrast to wild typepolymerase, incorporates dATP as efficiently as rATP in a single roundof phosphodiester bond formation.

[0060]FIG. 17A, FIG. 17B, and FIG. 17C—Detection of in vivo activitiesof N4 vRNAP and mini-vRNAP. Transcription of β-galactosidase α-peptideby fill size and mini-vRNAP was assayed on inducing-Xgal media (FIG.17A). Plasmid (PACYC) templates were constructed with a reporter gene(αpeptide of β-galactosidase) under the control of vRNAP promoter P2cloned in either of two orientations (FIG. 17B). Induction of mini-vRNAPled to production and accumulation of detectable levels of the protein,whereas full-length vRNAP was degraded (FIG. 17C).

[0061]FIG. 18—Examples of different monopartite target probes of theinvention.

[0062]FIG. 19—An example of a bipartite target probe of the invention.

[0063]FIG. 20—A basic embodiment of the invention for detecting a targetsequence using a bipartite target probe having target-complementarysequences that are contiguous when annealed to a target sequencecomprising a target nucleic acid or a target sequence tag that is joinedto an analyte-binding substance.

[0064]FIG. 21—An embodiment where a circular transcription substrate isused to obtain a multimeric transcription product by rolling circletranscription.

[0065]FIG. 22—An embodiment that uses coupled target-dependent rollingcircle replication and rolling circle transcription to amplify theamount of transcription product obtained. The copies of the targetsequence in the rolling circle replication product are identical to thetarget sequence in the sample and provide additional sites for annealingand ligation of bipartite target probes in order to obtain more circulartranscription substrates. Ligation of the bipartite target probecatenates the circular transcription substrate to the rolling circlereplication product comprising the replicated target sequence. Thecatenated circular transcription substrates must be released from therolling circle replication product to achieve efficient rolling circletranscription. The method for releasing the catenated circulartranscription substrates illustrated here is to include a quantity ofdUTP in the rolling circle replication reaction mix in addition to dTTPso that a dUMP residue is incorporated randomly about every 100-400nucleotides. Uracil-N-glycosylase and endonuclease IV, which cleave theDNA strand wherever dUMP is incorporated, is also included in thereaction mixture. Once the rolling circle replication product is cleavedso that, on average, most of the replicated target sequences are withinabout 150-200 nucleotides from a free 3′-end, the catenated circulartranscription substrates will be released during rolling circletranscription.

[0066]FIG. 23—An embodiment in which target-complementary sequences ofthe bipartite target probe are not contiguous when annealed to a targetsequence and the gap between the target-complementary sequences isfilled using simple target probes. In the embodiment illustrated here,the circular transcription substrate has a transcription terminationsequence so that only one copy of the transcription product is obtained,rather than a multimer of tandem oligomers as obtained from rollingcircle transcription.

[0067]FIG. 24—An embodiment in which target-complementary sequences ofthe bipartite target probe are not contiguous when annealed to a targetsequence and the gap between the target-complementary sequences isfilled by DNA polymerase extension.

[0068]FIG. 25—An embodiment for detecting a target sequence bygenerating a linear transcription substrate using monopartite targetprobes.

[0069]FIG. 26—A method for obtaining additional amplification oftranscription products. The method uses two bipartite target probescomprising single-stranded promoters to generate circular transcriptionsubstrates for rolling circle transcription, and reverse transcriptionof the resulting RNA products to make additional copies of sense oranti-sense target sequences for annealing and ligation of additionalfirst or second bipartite target probes, respectively, which in turn areused to transcribe more RNA, which is detected.

[0070]FIG. 27—A method for detecting a non-nucleic acid analyte using ananalyte-binding substance comprising an antibody that has acovalently—(e.g., chemically) or non-covalently—(e.g., using biotin andstreptavidin) attached target sequence tag comprising a target sequence.In the embodiment illustrated here, the signal for detection of theanalyte-binding substance and the analyte is generated by transcriptionof a circular transcription substrate obtained by annealing and ligationof a bipartite target probe. In this particular embodiment, the circulartranscription substrate has a transcription termination sequence so thatmultiple single RNA copies are obtained, rather than multimeric tandemcopies of an oligomeric RNA as obtained by rolling circle transcription.

[0071]FIG. 28—A method for detecting a non-nucleic acid analyte using ananalyte-binding substance that has a target sequence tag comprising atarget sequence, wherein the the signal for the analyte-bindingsubstance and the analyte is generated by rolling circle transcriptionof a circular transcription substrate obtained by annealing and ligationof a bipartite target probe. Since the target sequence tag is designedto have a size so that a free 3′-end is less than about 150 nucleotidesand preferably less than 50-100 nucleotides from the target sequence,the catenated circular transcription substrates are easily released fromthe target sequence tag. The analyte can be any of a broad range ofanalytes for which an analyte-binding substance is available or can beidentified.

DETAILED DESCRIPTION OF THE INVENTION

[0072] Methods are provided for using various deletion mutants ofbacteriophage N4-coded, virion RNA polymerases (mini-vRNAPs), which havebeen developed, cloned and expressed in E. coli, and purified in activeform. Mini-vRNAPs lack helicase-like activity. However, in the presenceof E. coli SSB protein, mini-vRNAPs efficiently transcribesingle-stranded DNA (ssDNA) transcription substrates that comprise asingle-stranded DNA target sequence as a template if the template isoperably joined to a sequence comprising a single-stranded 17-basepromoter. The enzymes efficiently incorporate derivatized nucleosidetriphosphates, and a specific amino acid mutant of mini-vRNAP (Y678F)also incorporates other nucleotides, such as 2′-deoxynucleosidetriphosphates, with greater efficiency. The present invention disclosesnovel methods, processes, compositions, and kits for amplifying anddetecting one or multiple target nucleic acid sequences in or from asample, including target sequences that differ by as little as onenucleotide. The target sequence or target sequences can comprise atleast a portion of one or more target nucleic acids comprising eitherRNA or DNA from any source, or a target sequence can comprise a targetsequence tag that is attached to an analyte-binding substance, such as,but not limited to, an antibody, thus permitting use of the methods andcompositions of the present invention to detect any analyte for whichthere is a suitable analyte-binding substance. The methods of theinvention involve obtaining transcription products using a transcriptionsubstrate as a template, wherein the transcription substrate is made byligating at least two different target-complementary sequencescomprising one or more target probes when the target-complementarysequences are annealed adjacently on the target sequence. Since thetarget sequence is required for annealing and ligation of thetarget-complementary sequences which make the transcription substrate,obtaining the transcription product is target-dependent. Therefore,detection of the transcription products is indicative of the presence ofthe target sequence comprising the target nucleic acid or the targetsequence tag joined to an analyte-binding substance in the sample. Theinvention will be understood from the description of the additionalbackground, compositions, processes, methods and kits herein below.

[0073] I. RNA Polymerases

[0074] a. Structure and Promoter Recognition of DNA-Dependent RNAPolymerases of the Prior Art

[0075] Inspection of the sequences of phage, archaebacterial,eubacterial, eukaryotic and viral DNA-dependent RNA polymerases hasrevealed the existence of two enzyme families. The eubacterial,eukaryotic, archaebacterial, chloroplast and the vaccinia virus RNApolymerases are complex multisubunit enzymes (5-14 subunits) composed oftwo large subunits, one to several subunits of intermediate molecularweight (30-50-kDa) and none to several subunits of small molecularweight (<30-kDa) (Archambault and Friesen, Microbiol. Rev. 57:703-724,1993; Record et al., Cell and Molecular Biology 1:792-821, 1995.Eubacterial RNA polymerases are the simplest with an β₂ββ′ corestructure. Sequence comparison of the genes coding for the differentsubunits of these enzymes has revealed: 1-sequence homology in eightsegments (A to H) between β′ and the largest subunit of other RNApolymerases, 2-sequence homology in nine segments (A to I) between β andthe next largest subunit of other RNA polymerases, 3-sequence homologyin 3 segments (1.1, 1.2 and 2) between a and a subunit in RNApolymerases I, II and III (Puhler, et al., Proc. Natl. Acad. Sci. USA86:4569-4573, 1989; Sweetser, et al., Proc. Natl. Acad. Sci. USA84:1192-1196, 1987). Not surprisingly, the crystal structures of yeastRNAP H and E. coli RNAP core revealed remarkable similarities (Zhang, etal., Cell 98:811-824, 1999; Cramer, et al., Sciencexpress, 19 Apr.,2001).

[0076] In contrast, members of the phage T7-like (T7, T3, SP6) family ofRNA polymerases consist of a single (˜100 kDa) polypeptide whichcatalyzes all functions required for accurate transcription (Cheetham,et al., Curr. Op. In Struc. Biol. 10:117-123, 2000). The heterodimericbacteriophage N4 RNAP II, nuclear-coded mitochondrial, and Arabidopsischloroplast RNA polymerases show sequence similarity to the phage RNApolymerases (Cermakian, et al., Nuc. Acids Res. 24:648-654, 1996;Hedtke, et al., Science 277:809-811, 1997; Zehring, et al., J. Biol.Chem. 258:8074-8080, 1983). Three sequence motifs-A and C, which containthe two aspartic acids required for catalysis, and motif B- areconserved in polymerases that use DNA as a template (Delarue, et al.,Protein Engineering 3:461-467, 1990). The crystal structure of T7 RNAPresembles a “cupped right hand” with “palm,” “fingers” and “thumb”subdomains (Sousa, et al., Nature 364:593-599, 1993). The two catalyticaspartates are present in the “palm” of the structure. This structure isshared by the polymerase domains of E. coli DNA polymerase I and HIVreverse transcriptase (Sousa, Trends in Biochem. Sci. 21:186-190, 1996).Genetic, biochemical and structural information indicates that T7 RNApolymerase contains additional structures dedicated to nascent RNAbinding, promoter recognition, dsDNA unwinding and RNA:DNA hybridunwinding (Cheetham, et al., Curr. Op. In Struc. Biol. 10:117-123, 2000;Sousa, Trends in Biochem. Sci. 21:186-190, 1996). This unwindingactivity of T7 RNAP and T7-like RNAPs is described in Japanese KokaiPatent No. Hei 4(1992)-304,900 as “helicase-like activity.”

[0077] Both Class I and Class II RNA polymerases recognize specificsequences, called promoters, on B form double-stranded DNA. Eubacterialpromoters (except those recognized by σ⁵⁴) are characterized by tworegions of sequence homology: the −10 and the −35 hexamers (Gross, etal., Cold Spring Harbor Symp. Quant. Biol. 63:141-156, 1998).Specificity of promoter recognition is conferred to the core enzyme bythe σ subunit, which makes specific interactions with the −10 and −35sequences through two distinct DNA binding domains (Gross, et al., ColdSpring Harbor Symp. Quant. Biol. 63:141-156, 1998). This modularpromoter structure is also present at the promoters for eukaryotic RNApolymerases I, II and III. Transcription factors TFIIIA and TFIIICdirect recognition of RNAP III to two separate sequences (boxes A and C,separated by defined spacing) at the 5S gene promoter, whiletranscription factors TFIIIB and TFIIIC direct recognition of thisenzyme to blocks A and B, separated by variable distance (31-74 bp) atthe tRNA promoters (Paule, et al., Nuc. Acids Res. 28:1283-1298, 2000).Sequences important for RNAP I transcription initiation at the humanrRNA promoters are also restricted to two regions: the “core” regionlocated at −40 to +1 and the “upstream” region present at −160 to −107(Paule, et al., Nuc. Acids Res. 28:1283-1298, 2000). Assembly of theinitiation complex at RNAP II promoters requires several generaltranscription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH).Recognition involves three core elements: the TATA box located atposition −30 and recognized by TBP, the initiator element located near−1, and the downstream promoter element near +30 (Roeder, TrendsBiochem. Sci. 21:327-335, 1996).

[0078] Promoters for the T7-like and mitochondrial RNAPs are simpler.The T7-type RNAP promoters span a continuous highly conserved 23 bpregion extending from position −17 to +6 relative to the start site oftranscription (+1) (Rong, et al., Proc. Natl. Acad. Sci. USA 95:515-519,1998). The yeast mitochondrial RNAP promoters are even smaller,extending from −8 to +1 (Shadel, et al., J. Biol. Chem. 268:16083-16086,1993). One exception are the promoters for N4 RNAP II, which arerestricted to two blocks of conserved sequence: a/tTTTA at +1 andAAGACCTG present 18-26 bp upstream of +1 (Abravaya, et al., J. Mol.Biol. 211:359-372, 1990).

[0079] The activity of the multisubunit class of RNA polymerases isenhanced by activators at weak promoters. Transcription activatorsgenerally bind at specific sites on double-stranded DNA upstream of the−35 region (with the exception of the T4 sliding clamp activator), or atlarge distances in the cases of enhancers (Sanders, et al., EMBO Journal16:3124-3132, 1997). Activators modulate transcription by increasing thebinding (formation of closed complex) or isomerization (formation ofopen complex) steps of transcription through interactions with the α orσ subunits of RNAP (Hochschild, et al., Cell 92:597-600, 1998). Anexception is N4SSB, the activator of E. Coli RNAPσ⁷⁰ at thebacteriophage N4 late promoters, which activates transcription throughdirect interactions with the β′ subunit of RNAP in the absence of DNAbinding (Miller, et al., Science 275:1655-1657, 1997).

[0080] Proteins that bind to ssDNAs with high affinity but withoutsequence specificity have been purified and characterized from severalprokaryotes, eukaryotes, and their viruses (Chase, et al., Ann. Rev.Biochem. 55:130-136, 1986). These proteins (SSBs), which are requiredfor replication, recombination and repair, bind stoichiometrically and,in many cases, cooperatively to ssDNA to cover the transientsingle-stranded regions of DNA that normally arise in vivo as a resultof replication, repair and recombination. Binding to DNA results in theremoval of hairpin structures found on ssDNA, providing an extendedconformation for proteins involved in DNA metabolism. Several lines ofevidence suggest that single-stranded DNA binding proteins play a moredynamic role in cellular processes. Genetic and biochemical evidenceindicates that these proteins are involved in a multitude ofprotein-protein interactions including transcription activation(Rothman-Denes, et al., Genes Devepmnt. 12:2782-2790, 1999).

[0081] b. The Bacteriophage N4 Virion RNA Polymerase and RNA PolymeraseEnzymes of the Present Invention

[0082] Bacteriophage N4 virion RNA polymerase (N4 vRNAP) is present inN4 virions and is injected into the E. coli cell at the beginning ofinfection, where it is responsible for transcription of the N4 earlygenes (Falco, et al., Proc. Natl. Acad. Sci. (USA) 74:520-523, 1977;Falco, et al., Virology 95:454-465, 1979; Malone, et al., Virology162:328-336, 1988). The N4 vRNAP gene maps to the late region of the N4genome (Zivin, et al., J. Mol. Biol. 152:335-356, 1981). N4 vRNAPpurified from virions is composed of a single polypeptide with anapparent molecular mass of approximately 320,000 kDa (Falco, et al.,Biol. Chem. 255:4339-4347, 1980). In contrast to other DNA-dependentRNAPases, N4 vRNAP recognizes promoters on single-stranded templates(Falco, et al., Proc. Natl. Acad. Sci. (USA) 75:3220-3224, 1978). Thesepromoters are characterized by conserved sequences and a 5 bp stem, 3base loop hairpin structure (FIG. 1) (Haynes, et al., Cell 41:597-605,1985; Glucksmann, et al., Cell 70:491-500, 1992). N4 vRNAP lacksunwinding or helicase-like activity on dsDNA and also lacks unwindingactivity on RNA:DNA hybrids. In vivo, E. coli gyrase and single-strandedbinding protein are required for transcription by N4 vRNAP (Falco, etal., J. Biol. Chem. 255:4339-4347, 1980; Markiewicz, et al., Genes andDev. 6:2010-2019, 1992).

[0083] Sequencing of the N4 vRNAP gene revealed an ORF coding for aprotein 3,500 amino acids in length (SEQ ID NO:1-2). Inspection of thesequence revealed no extensive homology to either the multisubunit orthe T7-like families of RNA polymerases. However, three motifs arepresent (FIG. 2A): the T/DxxGR motif found in DNA-dependent polymerases,and Motif B (Rx₃Kx₆₋₇YG), one of three motifs common to the Pol I andPol α DNA polymerases and the T7-like RNA polymerases.

[0084] C. Transcription Using N4 vRNAP Enzymes of the Present Invention

[0085] RNA synthesis requires RNA polymerase, a DNA template, anactivated precursor (the ribonucleoside triphosphates ATP, GTP, UTP andCTP (XTP)), and divalent metal ions such as Mg⁺² or Mn⁺². The metal ionMg⁺² is strongly preferred. Synthesis of RNA begins at the promoter siteon the DNA. This site contains a sequence which the RNA polymeraserecognizes and binds. The RNA synthesis proceeds until a terminationsite is reached. N4 vRNAP termination signals comprise a hairpin loopthat forms in the newly synthesized RNA which is followed by a string ofuracils (poly U). The sequence of the terminator signals for vRNAPpresent in the N4 genome include SEQ ID NOS:21-26. These N4 vRNAPtermination signals possess all of the characteristics of eubacterialsequence-dependent terminators. The ribonucleoside triphosphate may bederivatized with, for example, biotin. Derivatized XTPs can be used forthe preparation of derivatized RNA. Exemplary methods for makingderivatized XTPs are disclosed in detail in Rashtchian et al.,“Nonradioactive Labeling and Detection of Biomolecules,” C. Kessler,Ed., Springer-Verlag, N.Y., pp. 70-84, 1992, herein incorporated byreference.

[0086] Single-stranded DNA of varying lengths can be used as a templatefor RNA synthesis using the N4 vRNAP or mini-vRNAP. Oligonucleotides andpolynucleotides of intermediate length may be used. One particularsingle-stranded DNA that may be used is M13 DNA. M13 genomic DNA existstemporarily inside infected E. coli cells as a double-stranded DNAplasmid and is packaged as a small, single-stranded circular DNA intophage particles. M13 phage particles are secreted by an infected celland single-stranded DNA can be purified from these particles for use asa transcription template. Initially M13 phage vectors required a workingknowledge of phage biology and were primarily used for creatingsingle-strand DNA molecules for DNA sequencing. M13-derived cloningvectors called “phagemids” take advantage of M13 replication to producesingle-strand molecules, but can be propagated as conventionalColE1-based replicating double-strand plasmids.

[0087] EcoSSB is essential for N4 vRNAP transcription in vivo (Falco etal., Proc. Natl. Acad. Sci. (USA) 75:3220-3224, 1978; Glucksmann, etal., Cell 70:491-500, 1992). EcoSSB is a specific activator of N4 vRNAPon single-stranded and supercoiled double-stranded DNA templates.EcoSSB, unlike other SSBs, does not melt the N4 vRNAP promoter hairpinstructure (Glucksmann-Kuis, et al., Cell 84:147-154, 1996). EcoSSB has ahigh specificity for N4 vRNAP and mini-vRNAP resulting from EcoSSB'sability to stabilize the template-strand hairpin, whereas thenontemplate strand hairpin is destabilized. Other single-stranded DNAbinding proteins destabilize the template-strand hairpin(Glucksmann-Kuis, et al., Cell 84:147-154, 1996; Dai et al., GenesDevepmnt. 12:2782-2790, 1998). EcoSSB mediates template recycling duringtranscription by N4 vRNAP (Davidova, E K and Rothman-Denes, L B, Proc.Natl. Acad. Sci. USA 100:9250-9255, 2003, incorporated herein byreference). When EcoSSB is not used in N4 vRNAP transcription in vitro,a DNA:RNA hybrid is formed, preventing template reutilization. Withoutbeing bound by theory, it appears that EcoSSB functionally replaces theN-terminal domain that is present in T7 RNAP (but absent in N4 vRNAP)that is responsible for RNA binding and unwinding, resulting indisplacement of the RNA product from the template.

[0088] II. Genes and DNA Segments of RNA Polymerases of the PresentInvention

[0089] Important aspects of the present invention concern isolated DNAsegments and recombinant vectors encoding N4 vRNAP or more particularlymini-vRNAP or a mutant of mini-vRNAP and the creation and use ofrecombinant host cells through the application of DNA technology, thatexpress a wild type, polymorphic or mutant vRNAP. Other aspects of thepresent invention concern isolated nucleic acid segments and recombinantvectors encoding vRNAP. Sequences of SEQ ID NO:1, 3, 5, 7, 14 andbiologically functional equivalents thereof are used in the currentinvention. Single-stranded DNA oligonucleotides and polynucleotides canbe used as DNA templates.

[0090] The present invention concerns isolated nucleic acid segmentsthat are capable of expressing a protein, polypeptide or peptide thathas RNA polymerase activity. As used herein, the term “nucleic acidsegment” refers to a nucleic acid molecule that has been isolated freeof total genomic DNA of a particular species. Therefore, a nucleic acidsegment encoding vRNAP refers to a nucleic acid segment that containswild-type, polymorphic or mutant vRNAP coding sequences yet is isolatedaway from, or purified free from, total bacterial or N4 phage genomicDNA. Included within the term “nucleic acid segment,” are nucleic acidsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phage, viruses, andthe like.

[0091] Similarly, a nucleic acid segment comprising an isolated orpurified vRNAP gene refers to a nucleic acid segment including vRNAPprotein, polypeptide or peptide coding sequences and, in certainaspects, regulatory sequences, isolated substantially away from othernaturally occurring genes or protein encoding sequences. In thisrespect, the term “gene” is used for simplicity to refer to a functionalprotein, polypeptide or peptide encoding unit. As will be understood bythose of skill in the art, this functional term includes both genomicsequences, cDNA sequences and engineered segments that express, or maybe adapted to express, proteins, polypeptides, domains, peptides, vRNAPsand mutants of vRNAP encoding sequences.

[0092] “Isolated substantially away from other coding sequences” meansthat the gene of interest, in this case the vRNAP, or more particularlymini-vRNAP genes, forms the significant part of the coding region of thenucleic acid segment, and that the nucleic acid segment does not containlarge portions of naturally-occurring coding DNA, such as largechromosomal fragments or other functional genes or cDNA coding regions.Of course, this refers to the DNA segment as originally isolated, anddoes not exclude genes or coding regions later added to the segment bythe hand of man.

[0093] The term “a sequence essentially as set forth in SEQ ID NO:2means, for example, that the sequence substantially corresponds to aportion of SEQ ID NO:2 and has relatively few amino acids that are notidentical to, or a biologically functional equivalent of, the aminoacids of SEQ ID NO:2. This applies with respect to all peptide andprotein sequences herein, such as those of SEQ ID NO:4, 6, 8 and 15.

[0094] The term “biologically functional equivalent” is well understoodin the art and is further defined in detail herein. Accordingly,sequences that have about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or about 99%, and any range derivable therein, suchas, for example, about 70% to about 80%, and more preferably about 81%and about 90%; or even more preferably, between about 91% and about 99%;of amino acids that are identical or functionally equivalent to theamino acids of SEQ ID NO:2 will be sequences that are “essentially asset forth in SEQ ID NO:2,” provided the biological activity of theprotein is maintained. In particular embodiments, the biologicalactivity of a vRNAP protein, polypeptide or peptide, or a biologicallyfunctional equivalent, comprises transcription. A preferredtranscriptional activity that may be possessed by a vRNAP protein,polypeptide or peptide, or a biologically functional equivalent, is RNAsynthesis using single-stranded N4 vRNAP promoter-containing DNA as atemplate.

[0095] In certain other embodiments, the invention concerns isolatednucleic acid segments and recombinant vectors that include within theirsequence a nucleic acid sequence essentially as set forth in SEQ IDNO:1. The term “essentially as set forth in SEQ ID NO:1 is used in thesame sense as described above and means that the nucleic acid sequencesubstantially corresponds to a portion of SEQ ID NO:1 and has relativelyfew codons that are not identical, or functionally equivalent, to thecodons of SEQ ID NO:1. Again, nucleic acid segments that encodeproteins, polypeptide or peptides exhibiting RNAP activity will be mostpreferred.

[0096] The term “functionally equivalent codon” is used herein to referto codons that encode the same amino acid, such as the six codons forarginine and serine, and also refers to codons that encode biologicallyequivalent amino acids. For optimization of expression of vRNAP in humancells, the following codons are used, with preference of use from leftto right: Alanine Ala A GCC GCT GCA GCG; Cysteine Cys C TGC TGT;Aspartic acid Asp D GAG GAT; Glutamic acid Glu E GAG GAA; PhenylalaninePhe F TTC TTT; Glycine Gly G GGC GGG GGA GGT; Histidine His H CAC CAT;Isoleucine Ile I ATC ATT ATA; Lysine Lys K AAG AAA; Leucine Leu L CTGCTC TTG CTT CTA TTA; Methionine Met M ATG; Asparagine Asn N AAC AAT;Proline Pro P CCC CCT CCA CCG; Glutamine Gln Q CAG CAA; Arginine Arg RCGC AGG CGG AGA CGA CGT; Serine Ser S AGC TCC TCT AGT TCA TCG; ThreonineThr T ACC ACA ACT ACG; Valine Val V GTG GTC GTT GTA; Tryptophan Trp WTGG; Tyrosine Tyr Y TAC TAT. Thus, the most preferred codon for alanineis “GCC,” and the least is “GCG.” Codon usage for various organisms andorganelles can be found at the website http://www.kazusa.orjp/codon/,allowing one of skill in the art to optimize codon usage for expressionin various organisms using the disclosures herein. Thus, it iscontemplated that codon usage may be optimized for other animals, aswell as other organisms such as a prokaryote (e.g., an eubacteria), anarchaea, an eukaryote (e.g., a protist, a plant, a fungus, an animal), avirus and the like, as well as organelles that contain nucleic acids,such as mitochondria or chloroplasts, based on the preferred codon usageas would be known to those of ordinary skill in the art.

[0097] It will also be understood that amino acid and nucleic acidsequences may include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ sequences, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein, polypeptide or peptide activity. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

[0098] Excepting intronic or flanking regions, and allowing for thedegeneracy of the genetic code, sequences that have about 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99%, and anyrange derivable therein, such as, for example, about 50% to about 80%,and more preferably about 81% and about 90%; or even more preferably,between about 91% and about 99%; of nucleotides that are identical tothe nucleotides of SEQ ID NO:1 will be sequences that are “essentiallyas set forth in SEQ ID NO:1”.

[0099] a. Nucleic Acid Hybridization

[0100] The nucleic acid sequences disclosed herein also have a varietyof uses. Contiguous sequences from vRNAP nucleic acid sequences can beused, for example, as templates to synthesize vRNAP.

[0101] Naturally, the present invention also encompasses DNA segmentsthat are complementary, or essentially complementary, to the sequenceset forth in SEQ ID NO:1, 3, 5, 7 and 14. Nucleic acid sequences thatare “complementary” are those that are capable of base-pairing accordingto the standard Watson-Crick complementary rules. As used herein, theterm “complementary sequences” means nucleic acid sequences that arecomplementary, as may be assessed by the same nucleotide comparison setforth above, or as defined as being capable of hybridizing to thenucleic acid segment of SEQ ID NO:1 under stringent conditions such asthose described herein.

[0102] As used herein, a “DNA/RNA hybrid” is understood to mean that asingle strand of RNA is hybridized to a single strand of DNA.

[0103] The term “appropriate reaction conditions” as described hereinmean that temperature, pH, buffer, and other parameters are adjusted tooptimize the reaction rate and yield.

[0104] As used herein, “hybridization,” “hybridizes” or “capable ofhybridizing” is understood to mean the forming of a double or triplestranded molecule or a molecule with partial double or triple strandednature. The term “hybridization,” “hybridize(s)” or “capable ofhybridizing” encompasses the terms “stringent condition(s)” or “highstringency” and the terms “low stringency” or “low stringencycondition(s).”

[0105] As used herein “stringent condition(s)” or “high stringency” arethose conditions that allow hybridization between or within one or morenucleic acid strand(s) containing complementary sequence(s), butprecludes hybridization of random sequences. Stringent conditionstolerate little, if any, mismatch between a nucleic acid and a targetstrand. Such conditions are well known to those of ordinary skill in theart, and are preferred for applications requiring high selectivity.Non-limiting applications include isolating a nucleic acid, such as agene or a nucleic acid segment thereof, or detecting at least onespecific mRNA transcript or a nucleic acid segment thereof, and thelike.

[0106] Stringent conditions may comprise low salt and/or hightemperature conditions, such as provided by about 0.02 M to about 0.15 MNaCl at temperatures of about 50° C. to about 70° C. It is understoodthat the temperature and ionic strength of a desired stringency aredetermined in part by the length of the particular nucleic acid(s), thelength and nucleobase content of the target sequence(s), the chargecomposition of the nucleic acid(s), and to the presence or concentrationof formamide, tetramethylammonium chloride or other solvent(s) in ahybridization mixture.

[0107] It is also understood that these ranges, compositions andconditions for hybridization are mentioned by way of non-limitingexamples only, and that the desired stringency for a particularhybridization reaction is often determined empirically by comparison toone or more positive or negative controls. Depending on the applicationenvisioned it is preferred to employ varying conditions of hybridizationto achieve varying degrees of selectivity of a nucleic acid towards atarget sequence. In a non-limiting example, identification or isolationof a related target nucleic acid that does not hybridize to a nucleicacid under stringent conditions may be achieved by hybridization at lowtemperature and/or high ionic strength. For example, a medium stringencycondition could be provided by about 0.1 to 0.25 M NaCl at temperaturesof about 37° C. to about 55° C. Under these conditions, hybridizationmay occur even though the sequences of probe and target strand are notperfectly complementary, but are mismatched at one or more positions. Inanother example, a low stringency condition could be provided by about0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. toabout 55° C. Of course, it is within the skill of one in the art tofurther modify the low or high stringency conditions to suit aparticular application. For example, in other embodiments, hybridizationmay be achieved under conditions of 50 mM Tris-HCl (pH 8.3), 75 mM KCl,3 mM MgCl₂, 1.0 mM dithiothreitol, at temperatures between approximately20° C. to about 37° C. Other hybridization conditions utilized couldinclude approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 MM MgCl₂,at temperatures ranging from approximately 40° C. to about 72° C.

[0108] Accordingly, the nucleotide sequences of the disclosure may beused for their ability to selectively form duplex molecules withcomplementary stretches of genes or RNAs or to provide primers foramplification of DNA or RNA from tissues. Depending on the applicationenvisioned, it is preferred to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence.

[0109] The nucleic acid segments of the present invention, regardless ofthe length of the coding sequence itself, may be combined with other DNAsequences, such as promoters, enhancers, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol.

[0110] For example, nucleic acid fragments may be prepared that includea contiguous stretch of nucleotides identical to or complementary to SEQID NO:1, 3, 5, 7 or 14. Nucleic acid fragments for use as a DNAtranscription template may also be prepared. These fragments may beshort or of intermediate lengths, such as, for example, about 8, about10 to about 14, or about 15 to about 20 nucleotides, and that arechromosome-sized pieces, up to about 35,000, about 30,000, about 25,000,about 20,000, about 15,000, about 10,000, or about 5,000 base pairs inlength, as well as DNA segments with total lengths of about 1,000, about500, about 200, about 100 and about 50 base pairs in length (includingall intermediate lengths of these lengths listed above, i.e., any rangederivable therein and any integer derivable therein such a range) arealso contemplated to be useful.

[0111] For example, it will be readily understood that “intermediatelengths,” in these contexts, means any length between the quoted ranges,such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140,150, 160, 170, 180, 190, including all integers through the 200-500;500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; 5,000-10,000 ranges,up to and including sequences of about 12,001, 12,002, 13,001, 13,002,15,000, 20,000 and the like.

[0112] Various nucleic acid segments may be designed based on aparticular nucleic acid sequence, and may be of any length. By assigningnumeric values to a sequence, for example, the first residue is 1, thesecond residue is 2, etc., an algorithm defining all nucleic acidsegments can be created:

[0113] n to n+y

[0114] where n is an integer from I to the last number of the sequenceand y is the length of the nucleic acid segment minus one, where n+ydoes not exceed the last number of the sequence. Thus, for a 10-mer, thenucleic acid segments correspond to bases 1 to 10, 2 to 11, 3 to 12 . .. and/or so on. For a 15-mer, the nucleic acid segments correspond tobases 1 to 15, 2 to 16, 3 to 17 . . . and/or so on. For a 20-mer, thenucleic segments correspond to bases 1 to 20, 2 to 21, 3 to 22 . . .and/or so on. In certain embodiments, the nucleic acid segment may be aprobe or primer. As used herein, a “probe” generally refers to a nucleicacid used in a detection method or composition. As used herein, a“primer” generally refers to a nucleic acid used in an extension oramplification method or composition.

[0115] The use of a hybridization probe of between 17 and 100nucleotides in length, or in some aspect of the invention even up to 1-2Kb or more in length, allows the formation of a duplex molecule that isboth stable and selective. Molecules having complementary sequences overstretches greater than 20 bases in length are generally preferred, inorder to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of particular hybrid molecules obtained.One will generally prefer to design nucleic acid molecules havingcomplementary sequences over stretches of 20 to 30 nucleotides, or evenlonger where desired. Such fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means or byintroducing selected sequences into recombinant vectors for recombinantproduction.

[0116] In general, it is envisioned that the hybridization probesdescribed herein will be useful both as reagents in solutionhybridization, as in PCR, for detection of expression of correspondinggenes, as well as in embodiments employing a solid phase. In embodimentsinvolving a solid phase, the test DNA (or RNA) is adsorbed or otherwiseaffixed to a selected matrix or surface. This fixed, single-strandednucleic acid is then subjected to hybridization with selected probesunder desired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C content, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface to remove non-specificallybound probe molecules, hybridization is detected, or even quantified, bymeans of the label.

[0117] b. Nucleic Acid Amplification

[0118] Nucleic acid used as a template for amplification is isolatedfrom cells contained in the biological sample, according to standardmethodologies (Sambrook et al., In: Molecular Cloning: A LaboratoryManual 2 rev.ed., Cold Spring Harbor: Cold Spring Harbor LaboratoryPress, 1989). The nucleic acid may be genomic DNA or fractionated orwhole cell RNA. Where RNA is used, it may be desired to convert the RNAto a complementary DNA. In one embodiment, the RNA is whole cell RNA andis used directly as the template for amplification.

[0119] Pairs of primers that selectively hybridize to nucleic acids arecontacted with the isolated nucleic acid under conditions that permitselective hybridization. The term “primer,” as defined herein, is meantto encompass any nucleic acid that is capable of priming the synthesisof a nascent nucleic acid in a template-dependent process. Typically,primers are oligonucleotides from ten to twenty or thirty base pairs inlength, but longer sequences can be employed. Primers may be provided indouble-stranded or single-stranded form, although the single-strandedform is preferred.

[0120] Once hybridized, the nucleic acid:primer complex is contactedwith one or more enzymes that facilitate template-dependent nucleic acidsynthesis. Multiple rounds of amplification, also referred to as“cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

[0121] Next, the amplification product is detected. In certainapplications, the detection may be performed by visual means.Alternatively, the detection may involve indirect identification of theproduct via chemiluminescence, radioactive scintigraphy of incorporatedradiolabel or fluorescent label, or even via a system using electricalor thermal impulse signals (Affymax technology).

[0122] A number of template dependent processes are available to amplifythe marker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159.

[0123] Briefly, in PCR, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products, and the process isrepeated.

[0124] A reverse transcriptase PCR amplification procedure may beperformed in order to quantify the amount of mRNA amplified. Alternativemethods for reverse transcription utilize thermostable, RNA-dependentDNA polymerases. These methods are described in WO 90/07641, filed Dec.21, 1990. Polymerase chain reaction methodologies are well known in theart.

[0125] Another method for amplification is the ligase chain reaction(“LCR”), disclosed in EPA No. 320 308, incorporated herein by referencein its entirety. In LCR, two complementary probe pairs are prepared, andin the presence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

[0126] Q-beta Replicase, described in PCT Application No.PCT/US87/00880, incorporated herein by reference, may also be used asstill another amplification method in the present invention. In thismethod, a replicative sequence of RNA that has a region complementary tothat of a target is added to a sample in the presence of an RNApolymerase. The polymerase will copy the replicative sequence, which canthen be detected.

[0127] An isothermal amplification method, in which restrictionendonucleases and ligases are used to achieve the amplification oftarget molecules that contain nucleotide 5′-[alpha-thio]-triphosphatesin one strand of a restriction site may also be useful in theamplification of nucleic acids in the present invention.

[0128] Strand Displacement Amplification (SDA) is another method ofcarrying out isothermal amplification of nucleic acids which involvesmultiple rounds of strand displacement and synthesis, i e., nicktranslation. A similar method, called Repair Chain Reaction (RCR),involves annealing several probes throughout a region targeted foramplification, followed by a repair reaction in which only two of thefour bases are present. The other two bases can be added as biotinylatedderivatives for easy detection. A similar approach is used in SDA.Target specific sequences can also be detected using a cyclic probereaction (CPR). In CPR, a probe having 3′ and 5′ sequences ofnon-specific DNA and a middle sequence of specific RNA is hybridized toDNA that is present in a sample. Upon hybridization, the reaction istreated with RNase H, and the products of the probe identified asdistinctive products that are released after digestion. The originaltemplate is annealed to another cycling probe and the reaction isrepeated.

[0129] Still another amplification method described in GB ApplicationNo. 2 202 328, and in PCT Application No. PCT/US89/01025, each of whichis incorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

[0130] Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS), including nucleic acidsequence based amplification (NASBA) and 3SR (Gingeras et al., PCTApplication WO 88/10315). In NASBA, the nucleic acids can be preparedfor amplification by standard phenol/chloroform extraction, heatdenaturation of a clinical sample, treatment with lysis buffer andminispin columns for isolation of DNA and RNA or guanidinium chlorideextraction of RNA. These amplification techniques involve annealing aprimer which has target specific sequences. Following polymerization,DNA/RNA hybrids are digested with RNase H while double-stranded DNAmolecules are heat denatured again. In either case, the single-strandedDNA is made fully double-stranded by addition of second target specificprimer, followed by polymerization. The double-stranded DNA moleculesare then multiply transcribed by an RNA polymerase such as T7 or SP6. Inan isothermal cyclic reaction, the RNAs are reverse transcribed intosingle-stranded DNA, which is then converted to double-stranded DNA, andthen transcribed once again with an RNA polymerase such as T7 or SP6.The resulting products, whether truncated or complete, indicate targetspecific sequences.

[0131] Davey et al., EPA No. 329 822 disclose a nucleic acidamplification process involving cyclically synthesizing single-strandedRNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be usedin accordance with the present invention. The ssRNA is a template for afirst primer oligonucleotide, which is elongated by reversetranscriptase (RNA-dependent DNA polymerase). The RNA is then removedfrom the resulting DNA:RNA duplex by the action of ribonuclease H (RNaseH, an RNase specific for RNA in duplex with either DNA or RNA). Theresultant ssDNA is a template for a second primer, which also includesthe sequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

[0132] Miller et al., PCT Application WO 89/06700 disclose a nucleicacid sequence amplification scheme based on the hybridization of apromoter/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Thisscheme is not cyclic, i.e., new templates are not produced from theresultant RNA transcripts. Other amplification methods include “RACE”and “one-sided PCR” (Frohman, In: PCR Protocols: A Guide To Methods AndApplications, Academic Press, N.Y., 1990).

[0133] Methods based on ligation of two (or more) oligonucleotides inthe presence of nucleic acid having the sequence of the resulting“di-oligonucleotide,” thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention.

[0134] c. Nucleic Acid Detection

[0135] In certain embodiments, it will be advantageous to employ nucleicacid sequences of the present invention such as all or part of SEQ IDNO:1, 3, 5, 7, 14 or a mutant thereof in combination with an appropriatemeans, such as a label, for hybridization assays, RNase protection andNorthern hybridization. A wide variety of appropriate indicator meansare known in the art, including fluorescent, radioactive, enzymatic orother ligands, such as avidin/biotin, which are capable of beingdetected. In preferred embodiments, one may desire to employ afluorescent label or an enzyme tag such as urease, alkaline phosphataseor peroxidase, instead of radioactive or other environmentallyundesirable reagents. In the case of enzyme tags, colorimetric indicatorsubstrates are known that can be employed to provide a detection meansvisible to the human eye or spectrophotometrically, to identify specifichybridization with complementary nucleic acid-containing samples.

[0136] In embodiments wherein nucleic acids are amplified, it may bedesirable to separate the amplification product from the template andthe excess primer for the purpose of determining whether specificamplification has occurred. In one embodiment, amplification productsare separated by agarose, agarose-acrylamide or polyacrylamide gelelectrophoresis using standard methods (Sambrook et al., In: MolecularCloning: A Laboratory Manual 2 rev.ed., Cold Spring Harbor: Cold SpringHarbor Laboratory Press, 1989).

[0137] Alternatively, chromatographic techniques may be employed toeffect separation. There are many kinds of chromatography which may beused in the present invention: adsorption, partition, ion-exchange andmolecular sieve, and many specialized techniques for using themincluding column, paper, thin-layer and gas chromatography.

[0138] Amplification products must be visualized in order to confirmamplification of the marker sequences. One typical visualization methodinvolves staining of a gel with ethidium bromide and visualization underUV light. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theamplification products can then be exposed to x-ray film or visualizedunder the appropriate stimulating spectra, following separation.

[0139] In one embodiment, visualization is achieved indirectly.Following separation of amplification products, a labeled, nucleic acidprobe is brought into contact with the amplified marker sequence. Theprobe preferably is conjugated to a chromophore but may be radiolabeled.In another embodiment, the probe is conjugated to a binding partner,such as an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

[0140] In one embodiment, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art. Briefly,amplification products are separated by gel electrophoresis. The gel isthen contacted with a membrane, such as nitrocellulose, permittingtransfer of the nucleic acid and non-covalent binding. Subsequently, themembrane is incubated with a chromophore-conjugated probe that iscapable of hybridizing with a target amplification product. Detection isby exposure of the membrane to x-ray film or ion-emitting detectiondevices.

[0141] One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

[0142] Other methods for genetic screening to accurately detectmutations in genomic DNA, cDNA or RNA samples may be employed, dependingon the specific situation.

[0143] Historically, a number of different methods have been used todetect point mutations, including denaturing gradient gelelectrophoresis (“DGGE”), restriction enzyme polymorphism analysis,chemical and enzymatic cleavage methods, and others. The more commonprocedures currently in use include direct sequencing of target regionsamplified by PCR (see above) and single-strand conformation polymorphismanalysis (“SSCP”).

[0144] Another method of screening for point mutations is based on RNasecleavage of base pair mismatches in RNA/DNA and RNA/RNA heteroduplexes.As used herein, the term “mismatch” is defined as a region of one ormore unpaired or mispaired nucleotides in a double-stranded RNA/RNA,RNA/DNA or DNA/DNA molecule. This definition thus includes mismatchesdue to insertion/deletion mutations, as well as single and multiple basepoint mutations.

[0145] U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavageassay that involves annealing single-stranded DNA or RNA test samples toan RNA probe, and subsequent treatment of the nucleic acid duplexes withRNase A. After the RNase cleavage reaction, the RNase is inactivated byproteolytic digestion and organic extraction, and the cleavage productsare denatured by heating and analyzed by electrophoresis on denaturingpolyacrylamide gels. For the detection of mismatches, thesingle-stranded products of the RNase A treatment, electrophoreticallyseparated according to size, are compared to similarly treated controlduplexes. Samples containing smaller fragments (cleavage products) notseen in the control duplex are scored as positive.

[0146] Currently available RNase mismatch cleavage assays, includingthose performed according to U.S. Pat. No. 4,946,773, require the use ofradiolabeled RNA probes. Myers and Maniatis in U.S. Pat. No. 4,946,773describe the detection of base pair mismatches using RNase A. Otherinvestigators have described the use of an E. coli enzyme, RNase I, inmismatch assays. Because it has broader cleavage specificity than RNaseA, RNase I would be a desirable enzyme to employ in the detection ofbase pair mismatches if components can be found to decrease the extentof non-specific cleavage and increase the frequency of cleavage ofmismatches. The use of RNase I for mismatch detection is described inliterature from Promega Biotech. Promega markets a kit containing RNaseI that is shown in their literature to cleave three out of four knownmismatches, provided the enzyme level is sufficiently high.

[0147] The RNase Protection assay was first used to detect and map theends of specific mRNA targets in solution. The assay relies on beingable to easily generate high specific activity radiolabeled RNA probescomplementary to the mRNA of interest by in vitro transcription.Originally, the templates for in vitro transcription were recombinantplasmids containing bacteriophage promoters. The probes are mixed withtotal cellular RNA samples to permit hybridization to theircomplementary targets, then the mixture is treated with RNase to degradeexcess unhybridized probe. Also, as originally intended, the RNase usedis specific for single-stranded RNA, so that hybridized double-strandedprobe is protected from degradation. After inactivation and removal ofthe RNase, the protected probe (which is proportional in amount to theamount of target mRNA that was present) is recovered and analyzed on apolyacrylamide gel.

[0148] The RNase Protection assay was adapted for detection of singlebase mutations. In this type of RNase A mismatch cleavage assay,radiolabeled RNA probes transcribed in vitro from wild-type sequencesare hybridized to complementary target regions derived from testsamples. The test target generally comprises DNA (either genomic DNA orDNA amplified by cloning in plasmids or by PCR), although RNA targets(endogenous mRNA) have occasionally been used. If single nucleotide (orgreater) sequence differences occur between the hybridized probe andtarget, the resulting disruption in Watson-Crick hydrogen bonding atthat position (“mismatch”) can be recognized and cleaved in some casesby single-strand specific ribonuclease. To date, RNase A has been usedalmost exclusively for cleavage of single-base mismatches, althoughRNase I has recently been shown as useful also for mismatch cleavage.There are recent descriptions of using the MutS protein and otherDNA-repair enzymes for detection of single-base mismatches.

[0149] Nuclease S1 analysis of reaction products can be used to measureRNA. An exemplary procedure for S1 analysis involves hybridizationreaction with the RNA of interest (0.005-0.1 mg) and an excess of S1probe which comprises a labeled oligonucleotide complementary to 20-80or more sequential nucleotides of a specific RNA in S1 hybridizationbuffer (80% formamide, 0.4 M NaCl, 1 mM EDTA, 40 mM Pipes, pH 6.4).After denaturation for 4 min at 94° C., overnight hybridization at 30°C. and precipitation with ethanol, the S1 probe/RNA mixture isresuspended in S1 buffer (0.26 M NaCl, 0.05 M sodium acetate, pH 4.6,and 4.5 mM zinc sulfate). The sample is divided into two volumes and 100units of S1 nuclease (Sigma Chemical Company) is added to one tube. Thesamples are incubated for 60 minutes at 37° C.; then EDTA (10 mM finalconcentration) and 15 g polyl-polyC RNA are added and the sample isextracted with phenol/chloroform and precipitated in ethanol. Thesamples are then subjected to polyacrylamide gel electrophoresis.

[0150] One method to produce a radiolabeled RNA probe with high specificactivity includes admixing a radiolabeled NTP during transcription.Suitable isotopes for radiolabeling include ³⁵S- and ³²P-labeled UTP,GTP, CTP or ATP. For optimal results, a gel-purified radiolabeled RNAprobe which is preferentially 300-500 bases in length, with a specificactivity of 1-3×10 8 cpm/μg should be generated using the RNA polymeraseof the current invention. In order to produce this in vitro transcript,it is often advisable to use a high specific activity (e.g., [α-³²P]CTPat 3,000 Ci/mmol) NTP. To prevent background hybridization, it isimportant to remove plasmid template DNA by digestion which can be donewith, for example, RQ1 RNase-Free DNase followed byphenol:chloroform:isoamyl alcohol extraction and ethanol precipitation.

[0151] Another method for producing radiolabeled probes includes using ariboprobe system which can produce high specific activity, radiolabeledRNA probes or microgram quantities of in vitro transcript. Riboprobesare useful with radiolabeled RNA probes in many applications includingRNase protection, Northern hybridization, S1 analysis and in situhybridization assays. The principle components of an in vitrotranscription are the riboprobe, an RNA polymerase, a DNA template whichincludes a phage RNA polymerase promoter and ribonucleotidetriphosphates.

[0152] d. Cloning vRNAP Genes

[0153] The present invention contemplates cloning vRNAP, or moreparticularly mini-vRNAP genes. A technique often employed by thoseskilled in the art of protein production today is to obtain a so-called“recombinant” version of the protein, to express it in a recombinantcell and to obtain the protein, polypeptide or peptide from such cells.These techniques are based upon the “cloning” of a nucleic acid moleculeencoding the protein from a DNA library, i.e., on obtaining a specificDNA molecule distinct from other portions of DNA. This can be achievedby, for example, cloning a cDNA molecule, or cloning a genomic-like DNAmolecule.

[0154] The first step in such cloning procedures is the screening of anappropriate DNA library, such as, for example, from a phage, bacteria,yeast, fungus, mouse, rat, monkey or human. The screening protocol mayutilize nucleotide segments or probes that are designed to hybridize tocDNA or genomic sequences of vRNAPs from protists. Additionally,antibodies designed to bind to the expressed vRNAP proteins,polypeptides, or peptides may be used as probes to screen an appropriateviral, eubacterial, archaebacterial or eukaryotic DNA expressionlibrary. Alternatively, activity assays may be employed. The operationof such screening protocols are well known to those of skill in the artand are described in detail in the scientific literature, for example,in Sambrook et al., In: Molecular Cloning: A Laboratory Manual 2rev.ed., Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989,incorporated herein by reference. Moreover, as the present inventionencompasses the cloning of genomic segments as well as cDNA molecules,it is contemplated that suitable genomic cloning methods, as known tothose in the art, may also be used.

[0155] Encompassed by the invention are DNA segments encoding relativelysmall peptides, such as, for example, peptides of from about 8, about 9,about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, about 25, about 26, about 27, about 28, about 29, about 30,about 31, about 32, about 33, about 34, about 35, about 35, about 40,about 45, to about 50 amino acids in length, and more preferably, offrom about 15 to about 30 amino acids in length; as set forth in SEQ IDNO:2, 4, 6, 8 or 15 and also larger polypeptides up to and includingproteins corresponding to the full-length sequences set forth in SEQ IDNO:2 and SEQ ID NO:15, and any range derivable therein and any integerderivable in such a range. In addition to the “standard” DNA and RNAnucleotide bases, modified bases are also contemplated for use inparticular applications of the present invention.

[0156] III. Recombinant Vectors, Promoters, Host Cells and Expression

[0157] Recombinant vectors form an important further aspect of thepresent invention. The term “expression vector or construct” means anytype of genetic construct containing a nucleic acid coding for a geneproduct in which part or all of the nucleic acid encoding sequence iscapable of being transcribed. The transcript may be translated into aproteinaceous molecule, but it need not be, such as in the case ofmini-vRNAP transcribing an RNA using a single-stranded DNA template.Thus, in certain embodiments, expression includes both transcription ofa single-stranded DNA and translation of an RNA into the proteinproduct. In other embodiments, expression only includes transcription ofthe nucleic acid. A recombinant vector can also be used for delivery ofthe RNA of the current invention.

[0158] Particularly useful vectors are contemplated to be those vectorsin which the coding portion of the DNA segment, whether encoding a fulllength protein or smaller polypeptide or peptide, is positioned underthe transcriptional control of a promoter. A “promoter” refers to a DNAsequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. The phrases “operatively positioned,” “undercontrol” or “under transcriptional control” means that the promoter isin the correct location and orientation in relation to the nucleic acidto control RNA polymerase initiation and expression of the gene.

[0159] One particularly useful vector is pBAD. The pBAD expressionvectors allow for greater control of bacterial expression of recombinantproteins and allow tight regulation for turning expression on or off.pBAD vectors allow for dose dependent induction for modulation ofexpression levels. The pBAD expression system helps overcome two of themost common problems of heterologous protein expression in bacteria:toxicity of the recombinant protein to the host and insolubility of therecombinant protein when it is expressed at high, uncontrolled levels.In both cases, a tightly-regulated expression system is critical formaximizing recombinant protein yields. The pBAD expression system isbased on the araBAD operon which controls the arabinose metabolicpathway in E. coli and allows for precise modulation of heterologousexpression to levels that are optimal for recovering high yields of theprotein of interest (Guzman et al., J. Bact. 177:4121-4130, 1995).

[0160] a. Promoters

[0161] Any promoters normally found in a host cell in the native statecan be used in the present invention to drive expression of N4 vRNA ormini-vRNA polymerase. Also, promoters not normally found in the hostcell in the native state that are recognized by a native, normallynative host cell RNA polymerase, or non-native RNA polymerase expressedin the cell can be used in the present invention to drive expression ofthe RNA polymerase. Other promoters may be selected from a nucleic acidsequence database accessible to those of skill in the art, e.g.,GenBank, or the promoter can be isolated by a screening method. Apromoter recognized by the host cell can be operably linked to the geneor genes encoding the N4 RNA polymerase. The operable linkage can beconstructed using any known techniques for DNA manipulation, as referredto herein.

[0162] Promoters are described as either constitutive or inducible.Constitutive promoters actively drive expression of genes under theircontrol. Inducible promoters, in contrast, are activated in response tospecific environmental stimuli. Both constitutive and induciblepromoters can be used in the present invention for expressing non-hostgenes in a host cell.

[0163] Inducible promoters include, but are not limited to, trp, tac,lac, ara, reca, λPr, and λP1. These promoters and others that can beused in the present invention for expression of the N4 vRNA or mini-vRNApolymerase, in embodiments in which the host cell is E. coli, aredescribed by Makrides, Microbiological Reviews 60, 512-538, 1996.Further, in embodiments of the present invention wherein the host cellis a microbe other than E. coli, such as Saccharomyces, Bacillus, andPseudomonas, any inducible promoter known to those skilled in the art tobe active in the host cell can be used to drive expression of theheterologous RNA polymerase. (U.S. Pat. No. 6,218,145).

[0164] The promoter may be in the form of the promoter that is naturallyassociated with N4 vRNA or mini-vRNA polymerase, as may be obtained byisolating the 5′ non-coding sequences located upstream of the codingsegment or exon, for example, using recombinant cloning and/or PCRtechnology, in connection with the compositions disclosed herein (PCRtechnology is disclosed in U.S. Pat. No. 4,683,202 and U.S. Pat. No.4,682,195).

[0165] In other embodiments, it is contemplated that certain advantageswill be gained by positioning the coding DNA segment under the controlof a recombinant, or heterologous, promoter. As used herein, arecombinant or heterologous promoter is intended to refer to a promoterthat is not normally associated with N4 vRNA or mini-vRNA polymerase inits natural environment. Such promoters may include promoters normallyassociated with other genes, and/or promoters isolated from any otherbacterial, viral, eukaryotic, protist, or mammalian cell, and/orpromoters made by the hand of man that are not “naturally occurring,” ie., containing different elements from different promoters, or mutationsthat increase, decrease, or alter expression.

[0166] Naturally, it will be important to employ a promoter thateffectively directs the expression of the DNA segment in the cell type,organism, or even animal, chosen for expression. The use of promoter andcell type combinations for protein expression is generally known tothose of skill in the art of molecular biology, for example, seeSambrook et al. In: Molecular Cloning: A Laboratory Manual 2 rev.ed.,Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989,incorporated herein by reference. The promoters employed may beconstitutive, or inducible, and can be used under the appropriateconditions to direct high level expression of the introduced DNAsegment, such as is advantageous in the large-scale production ofrecombinant proteins, polypeptides or peptides.

[0167] At least one module in a promoter generally functions to positionthe start site for RNA synthesis. The best known example of this is theTATA box, but in some promoters lacking a TATA box, such as the promoterfor the mammalian terminal deoxynucleotidyl transferase gene and thepromoter for the SV40 late genes, a discrete element overlying the startsite itself helps to fix the place of initiation.

[0168] Additional promoter elements regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave been shown to contain functional elements downstream of the startsite as well. The spacing between promoter elements frequently isflexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinasepromoter, the spacing between promoter elements can be increased to 50base pairs apart before activity begins to decline. Depending on thepromoter, it appears that individual elements can function eitherco-operatively or independently to activate transcription.

[0169] The particular promoter that is employed to control theexpression of a nucleic acid is not believed to be critical, so long asit is capable of expressing the nucleic acid in the targeted cell. Thus,where a human cell is targeted, it is preferable to position the nucleicacid coding region adjacent to and under the control of a promoter thatis capable of being expressed in a human cell. Generally speaking, sucha promoter might include either a human or viral promoter.

[0170] In various other embodiments, the human cytomegalovirus (CMV)immediate early gene promoter, the SV40 early promoter and the Roussarcoma virus long terminal repeat can be used to obtain high-levelexpression of the instant nucleic acids. The use of other viral ormammalian cellular or bacterial phage promoters which are well-known inthe art to achieve expression are contemplated as well, provided thatthe levels of expression are sufficient for a given purpose.

[0171] In certain embodiments of the invention, promoter sequences maybe used that that are recognized specifically by a DNA-dependent RNApolymerase, such as, but not limited to, those described by Chamberlinand Ryan, In: The Enzymes. San Diego, Calif., Academic Press, 15:87-108,1982, and by Jorgensen et al., J. Biol. Chem. 266:645-655, 1991. Thesepromoters can be used to express a wild-type or mutant form of a miniVRNA polymerase of the invention. Several RNA polymerase promotersequences are especially useful, including, but not limited to,promoters derived from SP6 (e.g., Zhou and Doetsch, Proc. Nat. Acad.Sci. USA 90:6601-6605, 1993), T7 (e.g., Martin, and Coleman,Biochemistry 26:2690-2696, 1987) and T3 (e.g., McGraw et al., Nucl.Acid. Res. 13:6753-6766, 1985). An RNA polymerase promoter sequencederived from Thermus thermophilus can also be used (see, e.g., Wendt etal., Eur. J. Biochem. 191:467-472, 1990; Faraldo et al., J. Bact.174:7458-7462, 1992; Hartmann et al., Biochem. 69:1097-1104, 1987;Hartmann et al., Nucl. Acids Res. 19:5957-5964, 1991). The length of thepromoter sequence will vary depending upon the promoter chosen. Forexample, the T7 RNA polymerase promoter can be only about 25 bases inlength and act as a functional promoter, while other promoter sequencesrequire 50 or more bases to provide a functional promoter.

[0172] In other embodiments of the invention, a promoter is used that isrecognized by an RNA polymerase from a T7-like bacteriophage. Thegenetic organization of all T7-like phages that have been examined hasbeen found to be essentially the same as that of T7. Examples of T7-likephages according to the invention include, but are not limited toEscherichia coli phages T3, ΦI, ΦIII, W3 1, H, Y, A1, 122, cro, C21,C22, and C23; Pseudomonas putida phage gh-1; Salmonella typhimuriumphage SP6; Serratia marcescens phages IV; Citrobacter phage ViIII; andKlebsiella phage No. 11 (Hausmann, Current Topics in Microbiology andImmunology 75:77-109, 1976; Korsten et al., J. Gen. Virol. 43:57-73,1975; Dunn, et al., Nature New Biology 230:94-96, 1971; Towle, et al.,J. Biol. Chem. 250:1723-1733, 1975; Butler and Chamberlin, J. Biol.Chem. 257:5772-5778, 1982).

[0173] When a T7 RNA polymerase promoter, or another T7-like RNApolymerase promoter is used to express a wild-type or mutant form of agene for a miniV RNA polymerase of the invention, the gene can beexpressed in a host cell which expresses the T7 RNA polymerase, or thecorresponding T7-like RNA polymerase for the promoter used, wherein theRNA polymerase for the promoter is expressed either constitutively, ormore preferably, from an inducible promoter. By way of example, a T7 RNApolymerase expression system, such as, but not limited to, theexpression systems disclosed in, for example, U.S. Pat. Nos. 5,693,489and 5,869,320, the disclosures of which are incorporated herein byreference in their entirety.

[0174] b. Enhancers

[0175] Enhancers were originally detected as genetic elements thatincreased transcription from a promoter located at a distant position onthe same molecule of DNA. This ability to act over a large distance hadlittle precedent in classic studies of prokaryotic transcriptionalregulation. Subsequent work showed that regions of DNA with enhanceractivity are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins.

[0176] The basic distinction between enhancers and promoters isoperational. An enhancer region as a whole must be able to stimulatetranscription at a distance; this need not be true of a promoter regionor its component elements. On the other hand, a promoter must have oneor more elements that direct initiation of RNA synthesis at a particularsite and in a particular orientation, whereas enhancers lack thesespecificities. Promoters and enhancers are often overlapping andcontiguous, often seeming to have a very similar modular organization.

[0177] Additionally any promoter/enhancer combination (as per theEukaryotic Promoter Data Base EPDB, http://www.epd.isb-sib.ch/) couldalso be used to drive expression.

[0178] Eukaryotic cells can support cytoplasmic transcription fromcertain bacterial promoters if the appropriate bacterial polymerase isprovided, either as part of the delivery complex or as an additionalgenetic expression construct.

[0179] Turning to the expression of the proteinaceous molecules aftertranscription using the vRNAP, mini-vRNAP, or mutants thereof of thepresent invention, once a suitable clone or clones have been obtained,whether they be cDNA based or genomic, one may proceed to prepare anexpression system.

[0180] The engineering of DNA segment(s) for expression in a prokaryoticor eukaryotic system may be performed by techniques generally known tothose of skill in recombinant expression. It is believed that virtuallyany expression system may be employed in the expression of theproteinaceous molecules of the present invention.

[0181] Both cDNA and genomic sequences are suitable for eukaryoticexpression, as the host cell will generally process the genomictranscripts to yield functional mRNA for translation into proteinaceousmolecules. Generally speaking, it may be more convenient to employ asthe recombinant gene a cDNA version of the gene. It is believed that theuse of a cDNA version will provide advantages in that the size of thegene will generally be much smaller and more readily employed totransfect the targeted cell than will a genomic gene, which willtypically be up to an order of magnitude or more larger than the cDNAgene. However, it is contemplated that a genomic version of a particulargene may be employed where desired.

[0182] In expression, one will typically include a polyadenylationsignal to effect proper polyadenylation of the transcript. The nature ofthe polyadenylation signal is not believed to be crucial to thesuccessful practice of the invention, and any such sequence may beemployed. Preferred embodiments include the SV40 polyadenylation signaland the bovine growth hormone polyadenylation signal, convenient andknown to function well in various target cells. Also contemplated as anelement of the expression cassette is a terminator. These elements canserve to enhance message levels and to minimize read through from thecassette into other sequences.

[0183] c. Antisense, RNAi and Ribozymes

[0184] In some embodiments of the invention the vRNA polymerase can beused to synthesize antisense RNA, RNAi or interference RNA or ribozymes.

[0185] The term “antisense nucleic acid” is intended to refer to theoligonucleotides complementary to the base sequences of DNA and RNA.Antisense oligonucleotides, when introduced into a target cell,specifically bind to their target nucleic acid and interfere withtranscription, RNA processing, transport, translation, and/or stability.Targeting double-stranded (ds) DNA with oligonucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. An antisense nucleic acid may be complementary to SEQ IDNO:l, 3, 5, 7 or 14, complementary to a mini-vRNAP encoding sequence orto mini-vRNAP non-coding sequences. Antisense RNA constructs, or DNAencoding such antisense RNAs, may be employed to inhibit genetranscription or translation or both within a host cell, either in vitroor in vivo, such as within a host animal, including a human subject.

[0186] Antisense constructs may be designed to bind to the promoter andother control regions, exons, introns or even exon-intron boundaries(splice junctions) of a gene. It is contemplated that the most effectiveantisense constructs may include regions complementary to intron/exonsplice junctions. Thus, antisense constructs with complementary regionswithin 50-200 bases of an intron-exon splice junction may be used. Ithas been observed that some exon sequences can be included in theconstruct without seriously affecting the target selectivity thereof.The amount of exonic material included will vary depending on theparticular exon and intron sequences used. One can readily test whethertoo much exon DNA is included simply by testing the constructs in vitroto determine whether normal cellular function is affected or whether theexpression of related genes having complementary sequences is affected.

[0187] As stated above, “complementary” or “antisense” meanspolynucleotide sequences that are substantially complementary over theirentire length and have very few base mismatches. For example, sequencesof fifteen bases in length may be termed complementary when they havecomplementary nucleotides at thirteen or fourteen positions. Naturally,sequences which are completely complementary will be sequences which areentirely complementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme) could be designed. These molecules, though having lessthan 50% homology, would bind to target sequences under appropriateconditions.

[0188] It may be advantageous to combine portions of genomic DNA withcDNA or synthetic sequences to generate specific constructs. Forexample, where an intron is desired in the ultimate construct, a genomicclone will need to be used. The cDNA or a synthesized polynucleotide mayprovide more convenient restriction sites for the remaining portion ofthe construct and, therefore, would be used for the rest of thesequence.

[0189] While all or part of the gene sequence may be employed in thecontext of antisense construction, statistically, any sequence 17 baseslong should occur only once in the human genome and, therefore, sufficeto specify a unique target sequence. Although shorter oligomers areeasier to make and increase in vivo accessibility, numerous otherfactors are involved in determining the specificity of hybridization.Both binding affinity and sequence specificity of an oligonucleotide toits complementary target increases with increasing length. It iscontemplated that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more base pairs will be used. One can readilydetermine whether a given antisense nucleic acid is effective attargeting of the corresponding host cell gene simply by testing theconstructs in vivo to determine whether the endogenous gene's functionis affected or whether the expression of related genes havingcomplementary sequences is affected.

[0190] In certain embodiments, one may wish to employ antisenseconstructs which include other elements, for example, those whichinclude C-5 propyne pyrimidines. Oligonucleotides which contain C-5propyne analogues of uridine and cytidine have been shown to bind RNAwith high affinity and to be potent antisense inhibitors of geneexpression (Wagner et al., Science 260:1510-1513, 1993).

[0191] As an alternative to targeted antisense delivery, targetedribozymes may be used. The term “ribozyme” refers to an RNA-based enzymecapable of targeting and cleaving particular base sequences in oncogeneDNA and RNA. Ribozymes either can be targeted directly to cells, in theform of RNA oligonucleotides incorporating ribozyme sequences, orintroduced into the cell as an expression construct encoding the desiredribozymal RNA. Ribozymes may be used and applied in much the same way asdescribed for antisense nucleic acids. Sequences for ribozymes may beincluded in the DNA template to eliminate undesired 5′ end sequences inRNAs generated through T7 RNA polymerase transcription.

[0192] Ribozymes are RNA-protein complexes that cleave nucleic acids ina site-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, Proc. Natl. Acad. Sci. USA84:8788-8792, 1987; Gerlach et al., Nature 328:802-805, 1987; Forsterand Symons, Cell 49:211-220, 1987). For example, a large number ofribozymes accelerate phosphoester transfer reactions with a high degreeof specificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., Cell 27:487-496, 1981; Micheland Westhof, J. Mol. Biol. 216:585-610, 1990; Reinhold-Hurek and Shub,Nature 357:173-176, 1992). This specificity has been attributed to the.requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

[0193] Ribozyme catalysis has primarily been observed as part ofsequence specific cleavage/ligation reactions involving nucleic acids(Joyce, Nature 338:217-244, 1989; Cech et al., Cell 27:487-496, 1981).For example, U.S. Pat. No. 5,354,855 reports that certain ribozymes canact as endonucleases with a sequence specificity greater than that ofknown ribonucleases and approaching that of the DNA restriction enzymes.Thus, sequence-specific ribozyme-mediated inhibition of gene expressionmay be particularly suited to therapeutic applications (Scanlon et al.,Proc. Natl. Acad. Sci. USA 88:10591-10595, 1991; Sarver et al., Science247:1222-1225, 1990; Sioud et al., J. Mol. Biol. 223:831-835, 1992).Recently, it was reported that ribozymes elicited genetic changes insome cell lines to which they were applied; the altered genes includedthe oncogenes H-ras, c-fos and genes of HIV. Most of this work involvedthe modification of a target mRNA, based on a specific mutant codon thatis cleaved by a specific ribozyme. In light of the information includedherein and the knowledge of one of ordinary skill in the art, thepreparation and use of additional ribozymes that are specificallytargeted to a given gene will now be straightforward.

[0194] Several different ribozyme motifs have been described with RNAcleavage activity (reviewed in Symons, Annu. Rev. Biochem. 61:641-671,1992). Examples of ribozymes include sequences from the Group Iself-splicing introns including tobacco ringspot virus (Prody, et al.,Science 231:1577-1580, 1986), avocado sunblotch viroid (Palukaitis, etal., Virology 99:145-151, 1979; Symons, Nucl. Acids Res. 9:6527-6537,1981), and Lucerne transient streak virus (Forster and Symons, Cell49:211-220, 1987). Sequences from these and related viruses are referredto as hammerhead ribozymes based on a predicted folded secondarystructure.

[0195] Other suitable ribozymes include sequences from RNase P with RNAcleavage activity (Yuan, et al., Proc. Natl. Acad. Sci. USA89:8006-8010, 1992; Yuan and Altman, Science, 263:1269-1273, 1994),hairpin ribozyme structures (Berzal-Herranz, et al., Genes and Devel.6:129-134, 1992; Chowrira et al., Biochemistry 32:1088-1095, 1993) andhepatitis a virus based ribozymes (Perrotta and Been, Biochemistry31:16-21, 1992). The general design and optimization of ribozymedirected RNA cleavage activity has been discussed in detail (Haseloffand Gerlach, Nature 334:585-591, 1988; Symons, Annu. Rev. Biochem.61:641-671, 1992; Chowrira, et al., J. Biol. Chem. 269:25856-25864,1994; and Thompson, et al., Nature Medicine 1:277-278, 1995).

[0196] The other variable on ribozyme design is the selection of acleavage site on a given target RNA. Ribozymes are targeted to a givensequence by virtue of annealing to a site by complementary base pairinteractions. Two stretches of homology are required for this targeting.These stretches of homologous sequences flank the catalytic ribozymestructure defined above. Each stretch of homologous sequence can vary inlength from 7 to 15 nucleotides. The only requirement for defining thehomologous sequences is that, on the target RNA, they are separated by aspecific sequence which is the cleavage site. For hammerhead ribozymes,the cleavage site is a dinucleotide sequence on the target RNA, uracil(U) followed by either an adenine, cytosine or uracil (A, C or U;Perriman, et al., Gene 113:157-163, 1992; Thompson, et al., NatureMedicine 1:277-278, 1995). The frequency of this dinucleotide occurringin any given RNA is statistically 3 out of 16. Therefore, for a giventarget messenger RNA of 1000 bases, 187 dinucleotide cleavage sites arestatistically possible.

[0197] Designing and testing ribozymes for efficient cleavage of atarget RNA is a process well known to those skilled in the art. Examplesof scientific methods for designing and testing ribozymes are describedby Chowrira et al., J. Biol. Chem. 269:25856-25864 (1994) and Lieber andStrauss, Mol. Cell. Biol. 15: 540-551 (1995), each incorporated byreference. The identification of operative and preferred sequences foruse in ribozymes is simply a matter of preparing and testing a givensequence, and is a routinely practiced “screening” method known to thoseof skill in the art.

[0198] A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

[0199] d. Host Cells

[0200] Host cells may be derived from prokaryotes or eukaryotes,including yeast cells, insect cells, and mammalian cells, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded nucleic acid sequences. Numerous celllines and cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials (www.atcc.org). An appropriate host can be determinedby one of skill in the art based on the vector backbone and the desiredresult. A plasmid or cosmid, for example, can be introduced into aprokaryotic host cell for replication of many vector copies. Bacterialcells used as host cells for vector replication and/or expressioninclude DH5α, BL 21, JM109, and KC8, as well as a number of commerciallyavailable bacterial hosts such as SURE® Competent Cells and SOLOPACKGold Cells (STRATAGENE®, La Jolla, Calif.). Alternatively, bacterialcells such as E. coli LE392 could be used as host cells. Appropriateyeast cells include Saccharomyces cerevisiae, Saccharomyces pombe, andPichia pastoris.

[0201] Examples of eukaryotic host cells for replication and/orexpression of a vector include HeLa, NIH3T3, Jurrat, 293, Cos, CHO,Saos, BHK, C127 and PC12. Many host cells from various cell types andorganisms are available and would be known to one of skill in the art.Similarly, a viral vector may be used in conjunction with either aeukaryotic or prokaryotic host cell, particularly one that is permissivefor replication or expression of the vector.

[0202] Some vectors may employ control sequences that allow it to bereplicated and/or expressed in both prokaryotic and eukaryotic cells.One of skill in the art would further understand the conditions underwhich to incubate all of the above described host cells to maintain themand to permit replication of a vector. Also understood and known aretechniques and conditions that would allow large-scale production ofvectors, as well as production of the nucleic acids encoded by vectorsand/or their cognate polypeptides, proteins, or peptides.

[0203] It is proposed that vRNAP, or more particularly mini-vRNAP may beco-expressed with other selected proteinaceous molecules such as EcoSSBand other proteins of interest, wherein the proteinaceous molecules maybe co-expressed in the same cell or vRNAP gene may be provided to a cellthat already has another selected proteinaceous molecule. Co-expressionmay be achieved by co-transfecting the cell with two distinctrecombinant vectors, each bearing a copy of either of the respectiveDNAs. Alternatively, a single recombinant vector may be constructed toinclude the coding regions for both of the proteinaceous molecules,which could then be expressed in cells transfected with the singlevector. In either event, the term “co-expression” herein refers to theexpression of both the vRNAP gene and the other selected proteinaceousmolecules in the same recombinant cell.

[0204] As used herein, the terms “engineered” and “recombinant” cells orhost cells are intended to refer to a cell into which an exogenous DNAsegment or gene, such as a cDNA or gene encoding vRNAP, mini-vRNAP or amutant thereof, has been introduced. Therefore, engineered cells aredistinguishable from naturally occurring cells which do not contain arecombinantly introduced exogenous DNA segment or gene. Engineered cellsare thus cells having a gene or genes introduced through the hand ofman. Recombinant cells include those having an introduced cDNA orgenomic gene, and also include genes positioned adjacent to a promoternot naturally associated with the particular introduced gene.

[0205] To express a recombinant vRNAP, whether mutant or wild-type, inaccordance with the present invention one would prepare an expressionvector that comprises a wild-type, or mutant vRNAP proteinaceousmolecule-encoding nucleic acid under the control of one or morepromoters. To bring a coding sequence “under the control of” a promoter,one positions the 5′ end of the transcription initiation site of thetranscriptional reading frame generally between about 1 and about 50nucleotides “downstream” of the chosen promoter. The “upstream” promoterdirects transcription of the DNA and promotes expression of the encodedrecombinant protein, polypeptide or peptide. This is the meaning of“recombinant expression” in this context.

[0206] Many standard techniques are available to construct expressionvectors containing the appropriate nucleic acids andtranscriptional/translational control sequences in order to achieveprotein, polypeptide or peptide expression in a variety of hostexpression systems. Cell types available for expression include, but arenot limited to, bacteria, such as E. coli and B. subtilis, transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors.

[0207] Certain examples of prokaryotic hosts are E. coli strain RR1, E.coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E.coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325); bacilli such asBacillus subtilis; and other enterobacteriaceae such as Salmonellatyphimurium, Serratia marcescens, and various Pseudomonas species.

[0208] In general, plasmid vectors containing replicon and controlsequences which are derived from species compatible with the host cellare used in connection with these hosts. The vector ordinarily carries areplication origin, as well as marking sequences which are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is often transformed using derivatives of pBR322, a plasmid derivedfrom an E. coli species. pBR322 contains genes for ampicillin andtetracycline resistance and thus provides easy means for identifyingtransformed cells. The pBR plasmid, or other microbial plasmid or phagemust also contain, or be modified to contain, promoters which can beused by the microbial organism for expression of its own proteins.

[0209] In addition, phage vectors containing replicon and controlsequences that are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEM™-11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as E. coli LE392.

[0210] Further useful vectors include pIN vectors; and pGEX vectors, foruse in generating glutathione S-transferase (GST) soluble proteins forlater purification and separation or cleavage.

[0211] The following details concerning recombinant protein productionin bacterial cells, such as E. coli, are provided by way of exemplaryinformation on recombinant protein production in general, the adaptationof which to a particular recombinant expression system will be known tothose of skill in the art.

[0212] Bacterial cells, for example, E. coli, containing the expressionvector are grown in any of a number of suitable media, for example, LB.The expression of the recombinant proteinaceous molecule may be induced,e.g., by adding IPTG or any appropriate inducer to the media or byswitching incubation to a higher temperature, depending on the regulatedpromoter used. After culturing the bacteria for a further period,generally of between 2 and 24 hours, the cells are collected bycentrifugation and washed to remove residual media.

[0213] The bacterial cells are then lysed, for example, by disruption ina cell homogenizer, by sonication or cell press and centrifuged toseparate the dense inclusion bodies and cell membranes from the solublecell components. This centrifugation can be performed under conditionswhereby the dense inclusion bodies are selectively enriched byincorporation of sugars, such as sucrose, into the buffer andcentrifugation at a selective speed.

[0214] If the recombinant proteinaceous molecule is expressed in theinclusion bodies, as is the case in many instances, these can be washedin any of several solutions to remove some of the contaminating hostproteins, then solubilized in solutions containing high concentrationsof urea (e.g., 8M) or chaotropic agents such as guanidine hydrochloridein the presence of reducing agents, such as β-mercaptoethanol or DTT(dithiothreitol).

[0215] Under some circumstances, it may be advantageous to incubate theproteinaceous molecule for several hours under conditions suitable forthe proteinaceous molecule to undergo a refolding process into aconformation which more closely resembles that of the nativeproteinaceous molecule. Such conditions generally include lowproteinaceous molecule concentrations, less than 500 mg/ml, low levelsof reducing agent, concentrations of urea less than 2 M and often thepresence of reagents such as a mixture of reduced and oxidizedglutathione which facilitate the interchange of disulfide bonds withinthe proteinaceous molecule.

[0216] The refolding process can be monitored, for example, by SDS-PAGE,or with antibodies specific for the native molecule (which can beobtained from animals vaccinated with the native molecule or smallerquantities of recombinant proteinaceous molecule). Following refolding,the proteinaceous molecule can then be purified further and separatedfrom the refolding mixture by chromatography on any of several supportsincluding ion exchange resins, gel permeation resins or on a variety ofaffinity columns.

[0217] For expression in Saccharomyces, the plasmid YRp7, for example,is commonly used. This plasmid already contains the trp1 gene whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1. Thepresence of the trp1 lesion as a characteristic of the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

[0218] Suitable promoter sequences in yeast vectors include thepromoters for 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate protein, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. In constructingsuitable expression plasmids, the termination sequences associated withthese genes are also ligated into the expression vector downstream ofthe sequence desired to be expressed to provide polyadenylation of themRNA and termination.

[0219] In addition to micro-organisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. In addition to mammalian cells, these include insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus); and plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid) containing one or more RNAP coding sequences.

[0220] Different host cells have characteristic and specific mechanismsfor the post-translational processing and modification of proteinaceousmolecules. Appropriate cells lines or host systems can be chosen toensure the correct modification and processing of the foreignproteinaceous molecule expressed.

[0221] A number of viral-based expression systems may be utilized, forexample, commonly used promoters are derived from polyoma, Adenovirus 2,and most frequently Simian Virus 40 (SV40). The early and late promotersof SV40 virus are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication. Smaller or larger SV40 fragments may also be used,provided there is included the approximately 250 bp sequence extendingfrom the HindIII site toward the BglI site located in the viral originof replication.

[0222] In cases where an adenovirus is used as an expression vector, thecoding sequences may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1, E3, or E4)will result in a recombinant virus that is viable and capable ofexpressing an RNA in infected hosts.

[0223] Specific initiation signals may also be used for more efficienttranslation using the vRNAP of the current invention. These signalsinclude the ATG initiation codon and adjacent sequences. Exogenoustranslational control signals, including the ATG initiation codon, mayadditionally need to be provided. One of ordinary skill in the art wouldreadily be capable of determining this and providing the necessarysignals. It is well known that the initiation codon must be in-frame (orin-phase) with the reading frame of the desired coding sequence toensure translation of the entire insert. These exogenous translationalcontrol signals and initiation codons can be of a variety of origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of appropriate transcription enhancer elements andtranscription terminators.

[0224] In eukaryotic expression, one will also typically desire toincorporate into the transcriptional unit an appropriate polyadenylationsite (e.g., 5′-AATAAA-3′) if one was not contained within the originalcloned segment. Typically, the poly A addition site is placed about 30to 2000 nucleotides “downstream” of the termination site of theproteinaceous molecule at a position prior to transcription termination.

[0225] For long-term, high-yield production of a recombinant vRNAPprotein, polypeptide or peptide, stable expression is preferred. Forexample, cell lines that stably express constructs encoding a vRNAPprotein, polypeptide or peptide may be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with vectors controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines.

[0226] A number of selection systems may be used, including, but notlimited to, the herpes simplex virus thymidine kinase (tk),hypoxanthine-guanine phosphoribosyltransferase (hgprt) and adeninephosphoribosyltransferase (aprt) genes, in tk-, hgprt- or aprt-cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for dihydrofolate reductase (dhfr), that confers resistanceto methotrexate; gpt, that confers resistance to mycophenolic acid;neomycin (neo), that confers resistance to the aminoglycoside G-418; andhygromycin (hygro), that confers resistance to hygromycin.

[0227] Large scale suspension culture of bacterial cells in stirredtanks is a common method for production of recombinant proteinaceousmolecules. Two suspension culture reactor designs are in wide use—thestirred reactor and the airlift reactor. The stirred design hassuccessfully been used on an 8000 liter capacity for the production ofinterferon. Cells are grown in a stainless steel tank with aheight-to-diameter ratio of 1:1 to 3:1. The culture is usually mixedwith one or more agitators, based on bladed disks or marine propellerpatterns. Agitator systems offering less shear forces than blades havebeen described. Agitation may be driven either directly or indirectly bymagnetically coupled drives. Indirect drives reduce the risk ofmicrobial contamination through seals on stirrer shafts.

[0228] The airlift reactor for microbial fermentation relies on a gasstream to both mix and oxygenate the culture. The gas stream enters ariser section of the reactor and drives circulation. Gas disengages atthe culture surface, causing denser liquid free of gas bubbles to traveldownward in the downcomer section of the reactor. The main advantage ofthis design is the simplicity and lack of need for mechanical mixing.Typically, the height-to-diameter ratio is 10:1. The airlift reactorscales up relatively easily, has good mass transfer of gases andgenerates relatively low shear forces.

[0229] It is contemplated that the vRNAP proteins, polypeptides orpeptides of the invention may be “overexpressed,” i.e., expressed inincreased levels relative to its natural expression in cells. Suchoverexpression may be assessed by a variety of methods, includingradio-labeling and/or proteinaceous molecule purification. However,simple and direct methods are preferred, for example, those involvingSDS/PAGE and proteinaceous composition staining or western blotting,followed by quantitative analyses, such as densitometric scanning of theresultant gel or blot. A specific increase in the level of therecombinant protein, polypeptide or peptide in comparison to the levelin natural cells is indicative of overexpression, as is a relativeabundance of the specific proteinaceous molecule in relation to theother proteins produced by the host cell and, e.g., visible on a gel.

[0230] IV. Methods of Gene Transfer

[0231] In order to mediate the effect of transgene expression in a cell,it will be necessary to transfer the expression constructs (e.g., atherapeutic construct) of the present invention into a cell. Suchtransfer may employ viral or non-viral methods of gene transfer. Thissection provides a discussion of methods and compositions of gene ornucleic acid transfer, including transfer of antisense sequences.

[0232] The vRNAP genes are incorporated into a viral vector to mediategene transfer to a cell. Additional expression constructs encodingEcoSSB and other therapeutic agents as described herein may also betransferred via viral transduction using infectious viral particles, forexample, by transformation with an adenovirus vector of the presentinvention. Alternatively, a retrovirus, bovine papilloma virus, anadeno-associated virus (AAV), a lentiviral vector, a vaccinia virus, apolyoma virus, or an infective virus that has been engineered to expressa specific binding ligand may be used. Similarly, nonviral methods whichinclude, but are not limited to, direct delivery of DNA such as byinjection, electroporation, calcium phosphate precipitation, liposomemediated transfection, and microprojectile bombardment may be employed.Thus, in one example, viral infection of cells is used in order todeliver therapeutically significant genes to a cell. Typically, thevirus simply will be exposed to the appropriate host cell underphysiologic conditions, permitting uptake of the virus.

[0233] Microinjection can be used for delivery into a cell.Microinjection involves the insertion of a substance such as RNA into acell through a microelectrode. Typical applications include theinjection of drugs, histochemical markers (such as horseradishperoxidase or lucifer yellow) and RNA or DNA in molecular biologicalstudies. To extrude the substances through the very fine electrode tips,either hydrostatic pressure (pressure injection) or electric currents(ionophoresis) is employed.

[0234] V. Proteinaceous Compositions

[0235] In certain embodiments, the present invention concerns novelcompositions or methods comprising at least one proteinaceous molecule.The proteinaceous molecule may have a sequence essentially as set forthin SEQ ID NO:2, 4, 6, 8 or 15. The proteinaceous molecule may be a vRNAPor more preferably a mini-vRNAP, or a delivery agent. The proteinaceousmolecule may also be a mutated mini-vRNAP.

[0236] As used herein, a “proteinaceous molecule,” “proteinaceouscomposition,” “proteinaceous compound,” “proteinaceous chain” or“proteinaceous material” generally refers to, but is not limited to, aprotein of greater than about 200 amino acids or the full lengthendogenous sequence translated from a gene; a polypeptide of greaterthan about 100 amino acids; and/or a peptide of from about 3 to about100 amino acids. All the “proteinaceous” terms described above may beused interchangeably herein.

[0237] In certain embodiments the size of the at least one proteinaceousmolecule may comprise, but is not limited to, about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 31, about32, about 33, about 34, about 35, about 36, about 37, about 38, about39, about 40, about 41, about 42, about 43, about 44, about 45, about46, about 47, about 48, about 49, about 50, about 51, about 52, about53, about 54, about 55, about 56, about 57, about 58, about 59, about60, about 61, about 62, about 63, about 64, about 65, about 66, about67, about 68, about 69, about 70, about 71, about 72, about 73, about74, about 75, about 76, about 77, about 78, about 79, about 80, about81, about 82, about 83, about 84, about 85, about 86, about 87, about88, about 89, about 90, about 91, about 92, about 93, about 94, about95, about 96, about 97, about 98, about 99, about 100, about 110, about120, about 130, about 140, about 150, about 160, about 170, about 180,about 190, about 200, about 210, about 220, about 230, about 240, about250, about 275, about 300, about 325, about 350, about 375, about 400,about 425, about 450, about 475, about 500, about 525, about 550, about575, about 600, about 625, about 650, about 675, about 700, about 725,about 750, about 775, about 800, about 825, about 850, about 875, about900, about 925, about 950, about 975, about 1000, about 1100, about1200, about 1300, about 1400, about 1500, about 1750, about 2000, about2250, about 2500 or greater amino molecule residues, and any rangederivable therein.

[0238] As used herein, an “amino molecule” refers to any amino acid,amino acid derivative or amino acid mimic as would be known to one ofordinary skill in the art. In certain embodiments, the residues of theproteinaceous molecule are sequential, without any non-amino moleculeinterrupting the sequence of amino molecule residues. In otherembodiments, the sequence may comprise one or more non-amino moleculemoieties. In particular embodiments, the sequence of residues of theproteinaceous molecule may be interrupted by one or more non-aminomolecule moieties.

[0239] Accordingly, the term “proteinaceous composition” encompassesamino molecule sequences comprising at least one of the 20 common aminoacids in naturally synthesized proteins, or at least one modified orunusual amino acid, including but not limited to the following,beginning with the corresponding abbreviation: Aad 2-Aminoadipic acid;EtAsn N-Ethylasparagine; Baad 3-Aminoadipic acid; Hyl Hydroxylysine;Bala β-alanine, β-Amino-propionic acid; AHyl allo-Hydroxylysine; Abu2-Aminobutyric acid; 3Hyp 3-Hydroxyproline; 4Abu 4-Aminobutyric acid,piperidinic; 4Hyp 4-Hydroxy-proline acid; Acp 6-Aminocaproic acid; IdeIsodesmosine; Ahe 2-Amino-heptanoic acid; AIle allo-Isoleucine; Aib2-Aminoisobutyric acid; MeGly N-Methylglycine, sarcosine; Baib3-Aminoisobutyric acid; MeIle N-Methylisoleucine; Apm 2-Aminopimelicacid; MeLys 6-N-Methyllysine; Dbu 2,4-Diaminobutyric acid; MeValN-Methylvaline; Des Desmosine; Nva Norvaline; Dpm 2,2′-Diaminopimelicacid; Nle Norleucine; Dpr 2,3-Diaminopropionic acid; Orn Omithine EtGlyN-Ethylglycine.

[0240] In certain embodiments the proteinaceous composition comprises atleast one protein, polypeptide or peptide, such as vRNAP or mini-vRNAP.In further embodiments the proteinaceous composition comprises abiocompatible protein, polypeptide or peptide. As used herein, the term“biocompatible” refers to a substance which produces no significantuntoward effects when applied to, or administered to, a given organismaccording to the methods and amounts described herein. Such untoward orundesirable effects are those such as significant toxicity or adverseimmunological reactions. In preferred embodiments, biocompatibleprotein, polypeptide or peptide containing compositions will generallybe mammalian proteins or peptides or synthetic proteins or peptides eachessentially free from toxins, pathogens and harmful immunogens.

[0241] Proteinaceous compositions may be made by any technique known tothose of skill in the art, including the expression of proteins,polypeptides or peptides through standard molecular biologicaltechniques, the isolation of proteinaceous compounds from naturalsources, or the chemical synthesis of proteinaceous materials. Thenucleotide and protein, polypeptide and peptide sequences for variousgenes have been previously disclosed, and may be found at computerizeddatabases known to those of ordinary skill in the art. One such databaseis the National Center for Biotechnology Information's Genbank andGenPept databases (http://www.ncbi.nlm.nih.gov/). The coding regions forthese known genes may be amplified and/or expressed using the techniquesdisclosed herein or as would be know to those of ordinary skill in theart. Alternatively, various commercial preparations of proteins,polypeptides and peptides are known to those of skill in the art.

[0242] In certain embodiments, a proteinaceous compound may be purified.Generally, “purified” will refer to a specific or desired protein,polypeptide, or peptide composition that has been subjected tofractionation to remove various other proteins, polypeptides, orpeptides, and which composition substantially retains its activity, asmay be assessed, for example, by the protein assays, as would be knownto one of ordinary skill in the art for the specific or desired protein,polypeptide or peptide.

[0243] In certain embodiments, the proteinaceous composition maycomprise at least one antibody. A mini-vRNAP antibody may comprise allor part of an antibody that specifically recognizes mini-vRNAP. As usedherein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally,IgG and/or IgM are preferred because they are the most common antibodiesin the physiological situation and because they are most easily made ina laboratory setting.

[0244] The term “antibody” is used to refer to any antibody-likemolecule that has an antigen binding region, and includes antibodyfragments such as Fab′, Fab, F(ab′)₂, single domain antibodies (DABs),Fv, scFv (single chain Fv), and the like. The techniques for preparingand using various antibody-based constructs and fragments are well knownin the art. Means for preparing and characterizing antibodies are alsowell known in the art (See, e.g., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference).

[0245] It is contemplated that virtually any protein, polypeptide orpeptide containing component may be used in the compositions and methodsdisclosed herein. However, it is preferred that the proteinaceousmaterial is biocompatible. In certain embodiments, it is envisioned thatthe formation of a more viscous composition will be advantageous in thatthe high viscosity will allow the composition to be more precisely oreasily applied to the tissue and to be maintained in contact with thetissue throughout the procedure. In such cases, the use of a peptidecomposition, or more preferably, a polypeptide or protein composition,is contemplated. Ranges of viscosity include, but are not limited to,about 40 to about 100 poise. In certain aspects, a viscosity of about 80to about 100 poise is preferred.

[0246] Proteins and peptides suitable for use in this invention may beautologous proteins or peptides, although the invention is clearly notlimited to the use of such autologous proteins. As used herein, the term“autologous protein, polypeptide or peptide” refers to a protein,polypeptide or peptide which is derived or obtained from an organism.Organisms that may be used include, but are not limited to, a bovine, areptilian, an amphibian, a piscine, a rodent, an avian, a canine, afeline, a fungal, a plant, or a prokaryotic organism, with a selectedanimal or human subject being preferred. The “autologous protein,polypeptide or peptide” may then be used as a component of a compositionintended for application to the selected animal or human subject. Incertain aspects, the autologous proteins or peptides are prepared, forexample from whole plasma of the selected donor. The plasma is placed intubes and placed in a freezer at about −80° C. for at least about 12hours and then centrifuged at about 12,000 times g for about 15 minutesto obtain the precipitate. The precipitate, such as fibrinogen may bestored for up to about one year.

[0247] VI. Protein Purification

[0248] To prepare a composition comprising a vRNAP or mini-vRNAP, it isdesirable to purify the components or variants thereof Purification ofthe mini-vRNAP (SEQ ID NO:4) can be done in two step using affinitycolumns. The mini-vRNAP of SEQ ID NO:6 has been modified to comprise aHis tag such that purification can be done in a single step when usingmetal affinity columns such as those which employ nickel, cobalt orzinc. The full length vRNAP of SEQ ID NO:15 is also His tagged forpurification.

[0249] According to one embodiment of the present invention,purification of a peptide comprising vRNAP can be utilized ultimately tooperatively link this domain with a selective agent. Proteinpurification techniques are well known to those of skill in the art.These techniques involve, at one level, the crude fractionation of thecellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. A particularly efficient method of purifyingpeptides is affinity chromatography.

[0250] A tag may be used for protein or peptide purification anddetection such as hexahistidine (6-His, HHHHHH), FLAG (DYKDDDDK),hemaglutinin (HA, YPYDVPDYA) and c-myc (EQKLISEEDL). Other tags alsohave been generated, most of which are very small, comprising only a fewamino acids, and are therefore likely to have little to no effect on theconformation of the mature protein or peptide. These small tags do notrequire any special conformation to be recognized by antibodies. Systemsfor protein purification using these tags include NTA resin (6-His) orthe FLAG fusion system marketed by IBI (FLAG) where the fusion proteinis affinity-purified on an antibody column.

[0251] Certain aspects of the present invention concern thepurification, and in particular embodiments, the substantialpurification, of an encoded protein or peptide, such as a vRNAP. Theterm “purified protein or peptide” as used herein, is intended to referto a composition, isolatable from other components, wherein the proteinor peptide is purified to any degree relative to itsnaturally-obtainable state. A purified protein or peptide therefore alsorefers to a protein or peptide, free from the environment in which itmay naturally occur.

[0252] Generally, “purified” will refer to a protein or peptidecomposition, such as the vRNAP, that has been subjected to fractionationto remove various other components, and which composition substantiallyretains its expressed biological activity. Where the term “substantiallypurified” is used, this designation will refer to a composition in whichthe protein or peptide forms the major component of the composition,such as constituting about 50%, about 60%, about 70%, about 80%, about90%, about 95% or more of the proteins in the composition.

[0253] Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification” number. The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

[0254] Various techniques suitable for use in protein purification willbe well known to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

[0255] There is no general requirement that the protein or peptidealways be provided in their most purified state. Indeed, it iscontemplated that less substantially purified products will have utilityin certain embodiments. Partial purification may be accomplished byusing fewer purification steps in combination, or by utilizing differentforms of the same general purification scheme. For example, it isappreciated that a cation-exchange column chromatography performedutilizing an HPLC apparatus will generally result in a greater “-fold”purification than the same technique utilizing a low pressurechromatography system. Methods exhibiting a lower degree of relativepurification may have advantages in total recovery of protein product,or in maintaining the activity of an expressed protein.

[0256] It is known that the migration of a polypeptide can vary,sometimes significantly, with different conditions of SDS/PAGE. It willtherefore be appreciated that under differing electrophoresisconditions, the apparent molecular weights of purified or partiallypurified expression products may vary.

[0257] Ion exchange chromatography is a preferred method of separation.Using columns resins such as the metal affinity chromatography resinTALON are also preferred. TALON resin has an enhanced resolving powerfor polyhistidine-tagged proteins. This results in greater purity withless effort. TALON employs cobalt, an electropositive metal with aremarkably high affinity for polyhistidine-tagged proteins and a lowaffinity for other proteins. Often, no discernible binding of hostproteins occurs and a separate wash step is not required. The bindingproperties of cobalt allow protein elution under mild pH conditions thatprotect protein integrity.

[0258] Further concentration of the proteins can be done on an anionexchange column, such as the MonoQ column, a high resolution, anionexchange column. This column works at pressures less than 5 MPa, has ahigh capacity and gives very high chromatographic resolution.

[0259] High Performance Liquid Chromatography (HPLC) is characterized bya very rapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

[0260] Gel chromatography, or molecular sieve chromatography, is aspecial type of partition chromatography that is based on molecularsize. The theory behind gel chromatography is that the column, which isprepared with tiny particles of an inert substance that contain smallpores, separates larger molecules from smaller molecules as they passthrough or around the pores, depending on their size. As long as thematerial of which the particles are made does not adsorb the molecules,the sole factor determining rate of flow is the size. Hence, moleculesare eluted from the column in decreasing size, so long as the shape isrelatively constant. Gel chromatography is unsurpassed for separatingmolecules of different size because separation is independent of allother factors such as pH, ionic strength, temperature, etc. There alsois virtually no adsorption, less zone spreading and the elution volumeis related in a simple matter to molecular weight.

[0261] Affinity chromatography, a particularly efficient method ofpurifying peptides, is a chromatographic procedure that relies on thespecific affinity between a substance to be isolated and a molecule thatit can specifically bind to. This is a receptor-ligand type interaction.The column material is synthesized by covalently coupling one of thebinding partners to an insoluble matrix. The column material is thenable to specifically adsorb the substance from the solution. Elutionoccurs by changing the conditions to those in which binding will notoccur (e.g., alter pH, ionic strength, and temperature). Tags, asdescribed herein above, can be used in affinity chromatography.

[0262] The matrix should be a substance that itself does not adsorbmolecules to any significant extent and that has a broad range ofchemical, physical and thermal stability. The ligand should be coupledin such a way as to not affect its binding properties. The ligand alsoshould provide relatively tight binding, and it should be possible toelute the substance without destroying the sample or the ligand. One ofthe most common forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accordance with the present invention is discussed below.

[0263] An affinity column may have an N4 promoter which the vRNAP ormini-vRNAP proteins recognize attached to a matrix. This column would besuitable for use for the purification of polymerases with no additionaltags such as histidine tags.

[0264] VII. Separation, Quantitation, and Identification Methods

[0265] Following synthesis of the RNA, it may be desirable to separatethe amplification products of several different lengths from each otherand from the template and the excess primer.

[0266] a. Gel Electrophoresis

[0267] In one embodiment, amplification products are separated byagarose, agarose-acrylamide or polyacrylamide gel electrophoresis usingstandard methods.

[0268] b. Chromatographic Techniques

[0269] Alternatively, chromatographic techniques may be employed toeffect separation. There are many kinds of chromatography which may beused in the present invention: adsorption, partition, ion-exchange andmolecular sieve, and many specialized techniques for using themincluding column, paper, thin-layer and gas chromatography. In yetanother alternative, labeled cDNA products, such as biotin-labeled orantigen-labeled, can be captured with beads bearing avidin or antibody,respectively.

[0270] c. Microfluidic Techniques

[0271] Microfluidic techniques include separation on a platform such asmicrocapillaries, designed by ACLARA BioSciences Inc., or the LabChip™“liquid integrated circuits” made by Caliper Technologies Inc. Thesemicrofluidic platforms require only nanoliter volumes of sample, incontrast to the microliter volumes required by other separationtechnologies. Miniaturizing some of the processes involved in geneticanalysis has been achieved using microfluidic devices. For example,published PCT Application No. WO 94/05414, to Northrup and White reportsan integrated micro-PCR apparatus for collection and amplification ofnucleic acids from a specimen. U.S. Pat. Nos. 5,304,487 to Wilding etal., and 5,296,375 to Kricka et al., discuss devices for collection andanalysis of cell containing samples and are incorporated herein byreference. U.S. Pat. No. 5,856,174 describes an apparatus which combinesthe various processing and analytical operations involved in nucleicacid analysis and is incorporated herein by reference.

[0272] d. Capillary Electrophoresis

[0273] In some embodiments, it may be desirable to provide anadditional, or alternative means for analyzing the amplified genes. Inthese embodiments, micro capillary arrays are contemplated to be usedfor the analysis.

[0274] Microcapillary array electrophoresis generally involves the useof a thin capillary or channel which may or may not be filled with aparticular separation medium. Electrophoresis of a sample through thecapillary provides a size based separation profile for the sample.Microcapillary array electrophoresis generally provides a rapid methodfor size-based sequencing, PCR product analysis and restriction fragmentsizing. The high surface to volume ratio of these capillaries allows forthe application of higher electric fields across the capillary withoutsubstantial thermal variation across the capillary, consequentlyallowing for more rapid separations. Furthermore, when combined withconfocal imaging methods, these methods provide sensitivity in the rangeof attomoles, which is comparable to the sensitivity of radioactivesequencing methods. Typically, these methods comprise photolithographicetching of micron scale channels on a silica, silicon or othercrystalline substrate or chip, and can be readily adapted for use in thepresent invention. In some embodiments, the capillary arrays may befabricated from the same polymeric materials described for thefabrication of the body of the device, using the injection moldingtechniques described herein.

[0275] Rectangular capillaries are known as an alternative to thecylindrical capillary glass tubes. Some advantages of these systems aretheir efficient heat dissipation due to the large height-to-width ratioand, hence, their high surface-to-volume ratio and their high detectionsensitivity for optical on-column detection modes. These flat separationchannels have the ability to perform two-dimensional separations, withone force being applied across the separation channel, and with thesample zones detected by the use of a multi-channel array detector.

[0276] In many capillary electrophoresis methods, the capillaries, e.g.,fused silica capillaries or channels etched, machined or molded intoplanar substrates, are filled with an appropriate separation/sievingmatrix. Typically, a variety of sieving matrices are known in the artmay be used in the microcapillary arrays. Examples of such matricesinclude, e.g., hydroxyethyl cellulose, polyacrylamide, agarose and thelike. Generally, the specific gel matrix, running buffers and runningconditions are selected to maximize the separation characteristics ofthe particular application, e.g., the size of the nucleic acidfragments, the required resolution, and the presence of native orundenatured nucleic acid molecules. For example, running buffers mayinclude denaturants, chaotropic agents such as urea or the like, todenature nucleic acids in the sample.

[0277] e. Mass Spectroscopy

[0278] Mass spectrometry provides a means of “weighing” individualmolecules by ionizing the molecules in vacuo and making them “fly” byvolatilization. Under the influence of combinations of electric andmagnetic fields, the ions follow trajectories depending on theirindividual mass (m) and charge (z). For low molecular weight molecules,mass spectrometry has been part of the routine physical-organicrepertoire for analysis and characterization of organic molecules by thedetermination of the mass of the parent molecular ion. In addition, byarranging collisions of this parent molecular ion with other particles(e.g., argon atoms), the molecular ion is fragmented forming secondaryions by the so-called collision induced dissociation (CID). Thefragmentation pattern/pathway very often allows the derivation ofdetailed structural information. Other applications of massspectrometric methods known in the art can be found summarized inMethods in Enzymology, Vol. 193: “Mass Spectrometry” (J. A. McCloskey,editor), 1990, Academic Press, New York.

[0279] Due to the apparent analytical advantages of mass spectrometry inproviding high detection sensitivity, accuracy of mass measurements,detailed structural information by CID in conjunction with an MS/MSconfiguration and speed, as well as on-line data transfer to a computer,there has been considerable interest in the use of mass spectrometry forthe structural analysis of nucleic acids. The biggest hurdle to applyingmass spectrometry to nucleic acids is the difficulty of volatilizingthese very polar biopolymers. Therefore, “sequencing” had been limitedto low molecular weight synthetic oligonucleotides by determining themass of the parent molecular ion and through this, confirming thealready known sequence, or alternatively, confirming the known sequencethrough the generation of secondary ions (fragment ions) via CID in anMS/MS configuration utilizing, in particular, for the ionization andvolatilization, the method of fast atomic bombardment (FAB massspectrometry) or plasma desorption (PD mass spectrometry).

[0280] Two ionization/desorption techniques are electrospray/ionspray(ES) and matrix-assisted laser desorption/ionization (MALDI). As a massanalyzer, a quadrupole is most frequently used. The determination ofmolecular weights in femtomole amounts of sample is very accurate due tothe presence of multiple ion peaks, which all could be used for the masscalculation.

[0281] MALDI mass spectrometry, in contrast, can be particularlyattractive when a time-of-flight (TOF) configuration is used as a massanalyzer. Since, in most cases, no multiple molecular ion peaks areproduced with this technique, the mass spectra, in principle, looksimpler compared to ES mass spectrometry. DNA molecules up to amolecular weight of 410,000 Daltons could be desorbed and volatilized.More recently, the use of infra red lasers (IR) in this technique (asopposed to UV-lasers) has been shown to provide mass spectra of largernucleic acids such as synthetic DNA, restriction enzyme fragments ofplasmid DNA, and RNA transcripts up to a size of 2180 nucleotides.

[0282] In Japanese Patent No. 59-131909, an instrument is describedwhich detects nucleic acid fragments separated either byelectrophoresis, liquid chromatography or high speed gel filtration.Mass spectrometric detection is achieved by incorporating into thenucleic acids atoms which normally do not occur in DNA such as S, Br, Ior Ag, Au, Pt, Os, Hg.

[0283] f. Energy Transfer

[0284] Labeling hybridization oligonucleotide probes with fluorescentlabels is a well known technique in the art and is a sensitive,nonradioactive method for facilitating detection of probe hybridization.More recently developed detection methods employ the process offluorescence energy transfer (FET) rather than direct detection offluorescence intensity for detection of probe hybridization. FET occursbetween a donor fluorophore and an acceptor dye (which may or may not bea fluorophore) when the absorption spectrum of one (the acceptor)overlaps the emission spectrum of the other (the donor) and the two dyesare in close proximity. Dyes with these properties are referred to asdonor/acceptor dye pairs or energy transfer dye pairs. The excited-stateenergy of the donor fluorophore is transferred by a resonancedipole-induced dipole interaction to the neighboring acceptor. Thisresults in quenching of donor fluorescence. In some cases, if theacceptor is also a fluorophore, the intensity of its fluorescence may beenhanced. The efficiency of energy transfer is highly dependent on thedistance between the donor and acceptor, and equations predicting theserelationships have been developed. The distance between donor andacceptor dyes at which energy transfer efficiency is 50% is referred toas the Forster distance (Ro). Other mechanisms of fluorescence quenchingare also known including, for example, charge transfer and collisionalquenching.

[0285] Energy transfer and other mechanisms which rely on theinteraction of two dyes in close proximity to produce quenching are anattractive means for detecting or identifying nucleotide sequences, assuch assays may be conducted in homogeneous formats. Homogeneous assayformats are simpler than conventional probe hybridization assays whichrely on detection of the fluorescence of a single fluorophore label, asheterogeneous assays generally require additional steps to separatehybridized label from free label.

[0286] Homogeneous methods employing energy transfer or other mechanismsof fluorescence quenching for detection of nucleic acid amplificationhave also been described. Higuchi et al., Biotechnology 10:413-417(1992) disclose methods for detecting DNA amplification in real-time bymonitoring increased fluorescence of ethidium bromide as it binds todouble-stranded DNA. The sensitivity of this method is limited becausebinding of the ethidium bromide is not target specific and backgroundamplification products are also detected. WO 96/21144 disclosescontinuous fluorometric assays in which enzyme-mediated cleavage ofnucleic acids results in increased fluorescence. Fluorescence energytransfer is suggested for use in the methods, but only in the context ofa method employing a single fluorescent label which is quenched byhybridization to the target.

[0287] Signal primers or detector probes which hybridize to the targetsequence downstream of the hybridization site of the amplificationprimers have been described for use in detection of nucleic acidamplification (U.S. Pat. No. 5,547,861). The signal primer is extendedby the polymerase in a manner similar to extension of the amplificationprimers. Extension of the amplification primer displaces the extensionproduct of the signal primer in a target amplification-dependent manner,producing a double-stranded secondary amplification product which may bedetected as an indication of target amplification. The secondaryamplification products generated from signal primers may be detected bymeans of a variety of labels and reporter groups, restriction sites inthe signal primer which are cleaved to produce fragments of acharacteristic size, capture groups, and structural features such astriple helices and recognition sites for double-stranded DNA bindingproteins.

[0288] Many donor/acceptor dye pairs known in the art and may be used inthe present invention. These include, for example, fluoresceinisothiocyanate (FITC)/tetramethylrhodamine isothiocyanate (TRITC),FITC/Texas Red (Molecular Probes), FITC/N-hydroxysuccinimidyl1-pyrenebutyrate (PYB), FITC/eosin isothiocyanate (EITC),N-hydroxysuccinimidyl 1-pyrenesulfonate (PYS)/FITC, FITC/Rhodamine X,FITC/tetramethylrhodamine (TAMRA), and others. The selection of aparticular donor/acceptor fluorophore pair is not critical. For energytransfer quenching mechanisms, it is only necessary that the emissionwavelengths of the donor fluorophore overlap the excitation wavelengthsof the acceptor, i.e., there must be sufficient spectral overlap betweenthe two dyes to allow efficient energy transfer, charge transfer orfluorescence quenching. P-(dimethyl aminophenylazo) benzoic acid(DABCYL) is a non-fluorescent acceptor dye which effectively quenchesfluorescence from an adjacent fluorophore, e.g., fluorescein or5-(2′-aminoethyl) aminonaphthalene (EDANS). Any dye pair which producesfluorescence quenching in the detector nucleic acids of the inventionare suitable for use in the methods of the invention, regardless of themechanism by which quenching occurs. Terminal and internal labelingmethods are both known in the art and may be routinely used to link thedonor and acceptor dyes at their respective sites in the detectornucleic acid.

[0289] g. In vitro Studies

[0290] The synthesized RNA of the current invention may be used for invitro studies of spliceosome assembly, splicing reactions, or antisenseexperiments.

[0291] The spliceosome is a large, multisubunit complex consisting ofsmall, nuclear ribonucleoprotein particles (snRNPs). There are a totalof 5 snRNAs: U1, U2, U4, U5, and U6 which are small and uridine rich.Each snRNP has 1 or 2 of these RNAs. In addition to catalyzing thesplicing reaction, the spliceosome retains intermediate products,positions splice sites for precise joining of the exons, and preventsexons from diffusing away after cleavage and before ligation.Spliceosome catalysis involves concerted cleavage/ligation reactions inwhich the 2′-OH of branch site A attacks the 5′ splice site to form a2′-5′ phosphodiester bond with the first nucleotide of the intron. Theresulting 3′-OH at the end of the 5′ exon attacks the 3′ splice site torelease the lariat form of the intron and join the two exons togetherwith a normal 3′-5′ phosphodiester bond. At least 50 different proteinsare involved in spliceosome assembly and function. In the group I andgroup II introns, splicing is improved (in velocity and accuracy) byprotein factors.

[0292] VIII. Methods for Detecting a Target Sequence by Target-DependentTranscription

[0293] In one aspect, the present invention comprises novel methods,compositions and kits for amplifying, detecting and quantifying one ormultiple target nucleic acid sequences in a sample, including targetsequences that differ by as little as one nucleotide. The targetsequence or target sequences can comprise one or more target nucleicacids comprising either RNA or DNA from any source. The methods can alsobe used to detect an analyte of any type for which an analyte-bindingsubstance (such as, but not limited to, an antibody) can be obtained,provided that a tag comprising a target nucleic acid sequence is coupledor linked to said analyte-binding substance. The method is useful fordetecting specific nucleic acids or analytes in a sample with highspecificity and sensitivity. The method also has an inherently low levelof background signal. Preferred embodiments of the method consist of anannealing process, a DNA ligation process, an optional DNA polymeraseextension process, a transcription process, and, optionally, a detectionprocess. The DNA ligation joins a probe which has a firsttarget-complementary sequence and a single-stranded transcriptionpromoter for an RNA polymerase that lacks helicase-like activity toanother probe or another section of the same probe which has a secondtarget-complementary sequence and, optionally, a signal sequence. Thisstep is dependent on hybridization of the target-complementary probesequences to a target sequence and forms a transcription substrate forin vitro transcription of the second target-complementary sequence andthe signal sequence, if a signal sequence is present, in an amount thatis proportional to the amount of target sequence in the sample.

[0294] In vitro transcription amplifies the target-complementarysequence and the signal sequence, if present, in proportion to theamount of trancription substrate formed, permitting quantification ofthe amount of target sequence present. The invention comprises use of anovel RNA polymerase that lacks helicase-like activity and thatsynthesizes a transcription product using a single-strandedtranscription promoter and a single-stranded DNA template which isoperably or functionally joined or linked to the promoter using a methodof the invention. Joining of the promoter to the transcription templateto obtain a transcription substrate is target-dependent because joiningby ligation only occurs if the different target-complementary sequencescomprising the target probes are adjacent to or abut each when theyanneal to a target sequence, if the target sequence is present in thesample. The methods of the invention are therefore referred to herein as“target-dependent transcription.” A preferred RNA polymerase of theinvention is an N4 mini-vRNAP enzyme or a mutant form of an N4mini-vRNAP enzyme. A preferred RNA polymerase lacks a his tag or othertag sequence and comprises a transcriptionally active 1,106-amino aciddomain of the N4 vRNAP (herein designated “mini-vRNAP”) whichcorresponds to amino acids 998-2103 of N4 vRNAP. The RNA polymerasepreferentially has the amino sequence set forth in SEQ ID NO:4 or SEQ IDNO:6 or a mutant of the polymerase of SEQ ID NO:4 or SEQ ID NO:6, suchas a mutant with a mutation at position number Y678 or the polymerase ofSEQ ID NO:8. The vRNAP and mini-vRNA polymerase transcribe nucleic acidoperatively linked to an N4 promoter such as a P2 promoter of SEQ IDNO:16, SEQ ID NO:19, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29. Thepromoter of SEQ ID NO:16 or SEQ ID NO:28 is preferred.

[0295] The amount of transcription product obtained in a given reactiontime can also be increased using a coupled rolling circle replicationand rolling circle transcription reaction. The rolling circlereplication reaction uses a “target sequence amplification probe” (or a“TSA probe”) having target-complementary sequences at each end and anintervening sequence with a primer binding site. The TSA probe annealsto the target sequence, if present in the sample, and is ligated to forma “TSA circle.” After annealing a primer to the primer binding site,rolling circle replication of the TSA circle by a strand-displacing DNApolymerase under strand-displacing polymerization conditions generatesmultiple tandem copies the target sequence, which serve as annealing andligation sites for a bipartite target probe. After ligation of thebipartite target probe to form a circular transcription substrate,rolling circle transcription with an RNA polymerase of the presentinvention generates transcription products comprising multiple copies ofthe target sequence. A preferred RNA polymerase of the invention is anN4 mini-vRNAP enzyme or a mutant form of an N4 mini-vRNAP enzyme. Apreferred RNA polymerase lacks a his tag or other tag sequence andcomprises a transcriptionally active 1,106-amino acid domain of the N4vRNAP (herein designated “mini-vRNAP”) which corresponds to amino acids998-2103 of N4 vRNAP. The RNA polymerase preferentially has the aminosequence set forth in SEQ ID NO:4 or SEQ ID NO:6 or a mutant of thepolymerase of SEQ ID NO:4 or SEQ ID NO:6, such as a mutant with amutation at position number Y678 or the polymerase of SEQ ID NO:8. ThevRNAP and mini-vRNA polymerase transcribe nucleic acid operativelylinked to an N4 promoter such as a P2 promoter of SEQ ID NO:16, SEQ IDNO:19, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29. The promoter of SEQID NO:16 or SEQ ID NO:28 is preferred.

[0296] Yet another method for obtaining additional amplification of thetarget sequence is illustrated schematically in FIG. 26. This methodgenerates annealing and ligation sites for a second bipartite targetprobe by reverse transcription of the transcription products obtainedfollowing annealing of a first bipartite target probe to a targetsequence in the sample, ligation of the bipartite target probe, and invitro transcription of the resulting circular transcription substrate.

[0297] Following in vitro transcription, RNA complementary totarget-complementary probe sequences can be detected and quantifiedusing any of the conventional detection systems for nucleic acids suchas detection of fluorescent labels, enzyme-linked detection systems,antibody-mediated label detection, and detection of radioactive labels.Alternatively, the signal sequence in the transcription substrate cancomprise a sequence that is amplifiable and/or detectable by anothermethod. By way of example, but not of limitation, in some embodiments ofthe invention a signal sequence that encodes a substrate for an enzyme,such as, but not limited to, Q-beta replicase is used. In the latterembodiment, in vitro transcription of the ssDNA transcription substrateresults in synthesis of a substrate for a replicase, which is used torapidly and linearly amplify the signal further. Since the amplifiedproduct is directly proportional to the amount of target sequencepresent, quantitative measurements reliably represent the amount of atarget sequence in a sample. Major advantages of this method are thatthe ligation process, or an optional DNA polymerase extension, can bemanipulated to obtain single-nucleotide allelic discrimination, thetranscription process is isothermal, and signals are strictlyquantitative because the transcription reaction is linear and iscatalyzed by a highly processive enzyme, and signal amplification can beobtained which is also linear and greatly enhances the sensitivity of anassay or method. In multiplex assays, the transcription promotersequence used for in vitro transcription can be the same for all targetprobes.

[0298] A. Nucleic Acids and Polynucleotides of This Aspect of theInvention

[0299] A “nucleic acid” or “polynucleotide” of the invention is apolymer molecule comprising a series of “mononucleosides,” also referredto as “nucleosides,” in which the 3′-position of the pentose sugar ofone nucleoside is linked by an internucleoside linkage, such as, but notlimited to, a phosphodiester bond, to the 5′-position of the pentosesugar of the next nucleoside. A nucleoside linked to a phosphate groupis referred to as a “nucleotide.” The nucleotide that is linked to the5′-position of the next nucleotide in the series is referred to as “5′-of,” or “upstream of,” or the “5′-nucleotide” and the nucleotide that islinked to the 3′-position of said 5′ or upstream nucleotide is referredto as “3′- of,” or “downstream of,” or the “3′-nucleotide.” When twodifferent, non-overlapping polynucleotides or oligonucleotides hybridizeor anneal to different regions of the same linear complementary nucleicacid sequence, and the 3′-end of one polynucleotide or oligonucleotidepoints towards the 5′-end of the other, the former may be called the“upstream” polynucleotide or oligonucleotide and the latter the“downstream” polynucleotide or oligonucleotide.

[0300] The terms “3′- of” and “5′- of” are used herein to refer to theposition or orientation of a particular nucleic acid sequence or geneticelement encoded by a sequence, such as, but not limited to, atranscription promoter, relative to other sequences or genetic elements.

[0301] A “portion” or “region,” used interchangeably herein, of apolynucleotide or oligonucleotide is a contiguous sequence of 2 or morebases. In other embodiments, a region or portion is at least about anyof 3-5, 5-10, 10-15, 15-20, 20-25, 25-50, 50-100, 100-200, 200-400,400-600, 600-800, 800-1000, 1000-1500, or greater than 1500 contiguousnucleotides. As described above, a portion or region can be 5′- of or3′- of another portion or genetic element or sequence. A portion orregion can also comprise a 5′-end portion or a 3′-end portion, meaningit comprises a 5′-end or a 3′-end, respectively, or it can be a portionor region that is between a 5′-portion and a 3′-portion. Although acircular oligonucleotide or polynucleotide does not have an end or anend portion, it can have portions or regions that are 5′- of or 3′- ofanother portion or region or sequence or genetic element, which permitsorientation of one portion or region or sequence or genetic element withrespect to another within the circular nucleic acid strand.

[0302] Discussions of nucleic acid structure and synthesis aresimplified and clarified by adopting terms to name the two complementarystrands of a nucleic acid duplex. Traditionally, the strand encoding thesequences used to produce proteins or structural RNAs is designated asthe “plus” or “sense” strand, and its complement is designated as the“minus” or “anti-sense” strand. It is now known that in many cases, bothstrands are functional, and the assignment of the designation “plus” toone and “minus” to the other must then be arbitrary. Nevertheless, theterms are useful for designating the sequence orientation of nucleicacids or for designating the specific mRNA sequences transcribed and/orexpressed in a cell.

[0303] Those with knowledge in the art will understand these terms inthe context of nucleic acid chemistry and structure, particularlyrelated to the 3′- and 5′-positions of sugar moieties of canonicalnucleic acid nucleotides, and in the context of enzymatic synthesis ofnucleic acids in a 5′-to-3′ direction. Since most descriptions ofembodiments of the present invention are referring to single-strandednucleic acids, in most cases herein the inventors use the terms “3′- of”and “5′- of” to refer to the relative position or orientation of aparticular nucleic acid sequence or genetic element encoded by asequence that is located on the same nucleic acid strand. By way ofexample, a transcription promoter that is “3′- of the target sequence”refers to the position of a promoter relative to a target sequence onthe same strand. Those with knowledge in the art will understand that,if a first nucleic acid sequence is 3′- of a second sequence within onestrand, the complement of the first sequence will be 5′- of thecomplement of the second sequence in the complementary strand. Thedescription of the invention will be understood with respect to therelative position and orientation of a sequence or genetic elementwithin a particular strand, unless explicitly stated to the contrary.

[0304] The pentose sugar of the nucleic acid can be ribose, in whichcase, the nucleic acid or polynucleotide is referred to as “RNA,” or itcan be 2′-deoxyribose, in which case, the nucleic acid or polynucleotideis referred to as “DNA.” Alternatively, the nucleic acid can be composedof both DNA and RNA mononucleotides. In both RNA and DNA, each pentosesugar is covalently linked to one of four common “nucleic acid bases”(each also referred to as a “base”). Three of the predominantnaturally-occurring bases that are linked to the sugars (adenine,cytidine and guanine) are common for both DNA and RNA, while one base isdifferent; DNA has the additional base thymine, while RNA has theadditional base uridine. Those in the art commonly think of a smallpolynucleotide as an “oligonucleotide.” The term “oligonucleotide” asused herein is defined as a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably about 10 to 200nucleotides, but there is no defined limit to the length of anoligonucleotide. The exact size will depend on many factors, which inturn depends on the ultimate function or use of the oligonucleotide.

[0305] Also, for a variety of reasons, a nucleic acid or polynucleotideof the invention may comprise one or more modified nucleic acid bases,sugar moieties, or internucleoside linkages. By way of example, somereasons for using nucleic acids or polynucleotides that contain modifiedbases, sugar moieties, or internucleoside linkages include, but are notlimited to: (1) modification of the T_(m); (2) changing thesusceptibility of the polynucleotide to one or more nucleases; (3)providing a moiety for attachment of a label; (4) providing a label or aquencher for a label; or (5) providing a moiety, such as biotin, forattaching to another molecule which is in solution or bound to asurface.

[0306] In order to accomplish these or other goals, the invention doesnot limit the composition of the nucleic acids or polynucleotides of theinvention including any target probes, detection probes, such as, butnot limited to molecular beacons (U.S. Pat. Nos. 5,925,517 and 6,103,476of Tyagi et al. and U.S. Pat. No. 6,461,817 of Alland et al., all ofwhich are incorporated herein by reference); capture probes,oligonucleotides, or other nucleic acids used or detected in the assaysor methods, so long as each said nucleic acid functions for its intendeduse. By way of example, but not of limitation, the nucleic acid bases inthe mononucleotides may comprise guanine, adenine, uracil,-thymine, orcytidine, or alternatively, one or more of the nucleic acid bases maycomprise xanthine, allyamino-uracil, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl adenines, 2-propyl and other alkyl adenines,5-halouracil, 5-halo cytosine, 5-propynyl uracil, 5-propynyl cytosine,7-deazaadenine, 7-deazaguanine, 7-deaza-7-methyl-adenine,7-deaza-7-methyl-guanine, 7-deaza-7-propynyl-adenine,7-deaza-7-propynyl-guanine and other 7-deaza-7-alkyl or 7-aryl purines,N2-alkyl-guanine, N2-alkyl-2-amino-adenine, purine 6-aza uracil, 6-azacytosine and 6-aza thymine, 5-uracil (pseudo uracil), 4-thiouracil,8-halo adenine, 8-amino-adenine, 8-thiol adenine, 8-thiolalkyl adenines,8-hydroxyl adenine and other 8-substituted adenines and 8-halo guanines,8-amino-guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxylguanine and other 8-substituted guanines, other aza and deaza uracils,other aza and deaza thymidines, other aza and deaza cytosine, aza anddeaza adenines, aza and deaza guanines or 5-trifluoromethyl uracil and5-trifluorocytosine. Still further, they may comprise a nucleic acidbase that is derivatized with a biotin moiety, a digoxigenin moiety, afluorescent or chemiluminescent moiety, a quenching moiety or some othermoiety. The invention is not limited to the nucleic acid bases listed;this list is given to show the broad range of bases which may be usedfor a particular purpose in a method.

[0307] When a molecule comprising both a nucleic acid and a peptidenucleic acid (PNA) is used in the invention, modified bases can be usedin one or both parts. For example, binding affinity can be increased bythe use of certain modified bases in both the nucleotide subunits thatmake up the 2′-deoxyoligonucleotides of the invention and in the peptidenucleic acid subunits. Such modified bases may include5-propynylpyrimidines, 6-azapyrimidines, and N-2, N-6 and O-6substituted purines including 2-aminopropyladenine. Other modifiedpyrimidine and purine base are also expected to increase the bindingaffinity of macromolecules to a complementary strand of nucleic acid.

[0308] With respect to nucleic acids or polynucleotides of theinvention, one or more of the sugar moieties can comprise ribose or2′-deoxyribose, or alternatively, one or more of the sugar moieties canbe some other sugar moiety, such as, but not limited to,2′-fluoro-2′-deoxyribose or 2′-O-methyl-ribose, which provide resistanceto some nucleases.

[0309] The internucleoside linkages of nucleic acids or polynucleotidesof the invention can be phosphodiester linkages, or alternatively, oneor more of the internucleoside linkages can comprise modified linkages,such as, but not limited to, phosphorothioate, phosphorodithioate,phosphoroselenate, or phosphorodiselenate linkages, which are resistantto some nucleases.

[0310] A variety of methods are known in the art for making nucleicacids having a particular sequence or that contain particular nucleicacid bases, sugars, internucleoside linkages, chemical moieties, andother compositions and characteristics. Any one or any combination ofthese methods can be used to make a nucleic acid, polynucleotide, oroligonucleotide for the present invention. The methods include, but arenot limited to: (1) chemical synthesis (usually, but not always, using anucleic acid synthesizer instrument); (2) post-synthesis chemicalmodification or derivatization; (3) cloning of a naturally occurring orsynthetic nucleic acid in a nucleic acid cloning vector (e.g., seeSambrook, et al., Molecular Cloning: A Laboratory Approach SecondEdition, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. and Sambrook and Russell, Molecular Cloning, A Laboratory Manual,Third Edition, 2001, Cold Spring Harbor Laboratory Press,) such as, butnot limited to a plasmid, bacteriophage (e.g., M13 or lamba), phagemid,cosmid, fosmid, YAC, or BAC cloning vector, including vectors forproducing single-stranded DNA; (4) primer extension using an enzyme withDNA template-dependent DNA polymerase activity, such as, but not limitedto, Klenow, T4, T7, rBst, Taq, Tfl, or Tth DNA polymerases, includingmutated, truncated (e.g., exo-minus), or chemically-modified forms ofsuch enzymes; (5) PCR (e.g., see Dieffenbach, C. W., and Dveksler, eds.,PCR Primer: A Laboratory Manual, 1995, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.); (6) reverse transcription (includingboth isothermal synthesis and RT-PCR) using an enzyme with reversetranscriptase activity, such as, but not limited to, reversetranscriptases derived from avian myeloblasosis virus (AMV), Maloneymurine leukemia virus (MMLV), Bacillus stearothermophilis (rBst), orThermus thermophilus (Tth); (7) in vitro transcription using an enzymewith RNA polymerase activity, such as, but not limited to, SP6, T3, orT7 RNA polymerase, Tth RNA polymerase, E. coli RNA polymerase, or SP6 orT7 R&DNA™ Polymerase (Epicentre Technologies, Madison, Wis., USA), oranother enzyme; (8) use of restriction enzymes and/or modifying enzymes,including, but not limited to exo- or endonucleases, kinases, ligases,phosphatases, methylases, glycosylases, terminal transferases, includingkits containing such modifying enzymes and other reagents for makingparticular modifications in nucleic acids; (9) use of polynucleotidephosphorylases to make new randomized nucleic acids; (10) othercompositions, such as, but not limited to, a ribozyme ligase to join RNAmolecules; and/or (11) any combination of any of the above or othertechniques known in the art. Oligonucleotides and polynucleotides,including chimeric (i.e., composite) molecules and oligonucleotides withmodified bases, sugars, or internucleoside linkages are commerciallyavailable (e.g., TriLink Biotechnologies, San Diego, Calif., USA orIntegrated DNA Technologies, Coralville, Iowa).

[0311] The terms “hybridize” or “anneal” and “hybridization” or“annealing” refer to the formation of complexes between nucleotidesequences on opposite or complementary nucleic acid strands that aresufficiently complementary to form complexes via Watson-Crick basepairing. Where a target probe, primer, transcription substrate, oranother oligonucleotide or polynucleotide “hybridizes” or “anneals” withtarget nucleic acid or a template or another oligonucleotide orpolynucleotide, such complexes or “hybrids” are sufficiently stable toserve the function required for ligation, DNA polymerase extension, orother function for which it is intended. *

[0312] With respect to nucleic acid synthesis, a “template” is a nucleicacid molecule that is being copied by a nucleic acid polymerase. Thesynthesized copy is complementary to the template. Both RNA and DNA arealways synthesized in the 5′-to-3′ direction and the two strands of anucleic acid duplex always are aligned so that the 5′-ends of the twostrands are at opposite ends of the duplex (and, by necessity, so thenare the 3′-ends). In general, DNA polymerases, including bothDNA-dependent (i.e, having a DNA template) and RNA-dependent (i.e.,having an RNA template, which enzyme is also called a “reversetranscriptase”) DNA polymerases, require a primer for synthesis of DNA.In general, RNA polymerases do not require a primer for RNA synthesis.

[0313] With respect to ligation, a “template” or a “ligation template”or a “template for ligation” is a nucleic acid molecule to which two ormore complementary oligonucleotides, target probes, or other nucleicacids that are to be ligated anneal or hybridize prior to ligation,wherein the ends of said nucleic acid molecules that are to be ligatedare adjacent to each other when annealed to the ligation template.

[0314] B. Samples, Analytes and Target Nucleic Acids of This Aspect ofthe Invention

[0315] A “sample” or a “biological sample” according to the presentinvention is used in its broadest sense. A sample is any specimen thatis collected from or is associated with a biological or environmentalsource, or which comprises or contains biological material, whether inwhole or in part, and whether living or dead. In some embodiments of theinvention a sample can also be chemically synthesized or derived in thelaboratory, rather than from a natural source.

[0316] Biological samples may be plant or animal, including human, fluid(e.g., blood or blood fractions, urine, saliva, sputum, cerebral spinalfluid, pleural fluid, milk, lymph, or semen), swabs (e.g., buccal orcervical swabs), solid (e.g., stool), microbial cultures (e.g., plate orliquid cultures of bacteria, fungi, parasites, protozoans, or viruses),or cells or tissue (e.g., fresh or paraffin-embedded tissue sections,hair follicles, mouse tail snips, leaves, or parts of human, animal,plant, microbial, viral, or other cells, tissues, organs or wholeorganisms, including subcellular fractions or cell extracts), as well asliquid and solid food and feed products and ingredients such as dairyitems, vegetables, meat and meat by-products, and waste. Biologicalsamples may be obtained from all of the various families of domesticplants or animals, as well as wild animals or plants.

[0317] Environmental samples include environmental material such assurface matter, soil, water, air, or industrial samples, as well assamples obtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items. These examplesare not to be construed as limiting the sample types applicable to thepresent invention.

[0318] In short, a sample comprises a specimen from any source thatcontains or may contain a target nucleic acid.

[0319] A sample on which the assay method of the invention is carriedout can be a raw specimen of biological material, such as serum or otherbody fluid, tissue culture medium or food material. More typically, themethod is carried out on a sample that is a processed specimen, derivedfrom a raw specimen by various treatments to remove materials that wouldinterfere with detection of a target nucleic acid or an amplificationproduct thereof. Methods for processing raw samples to obtain a samplemore suitable for the assay methods of the invention are well known inthe art.

[0320] An “analyte” means a substance or a part of a substance whosepresence, concentration or amount in a sample is being determined in anassay. An analyte is sometimes referred to as a “target substance” or a“target molecule” or a “target analyte” of an assay. An analyte may alsobe referred to more specifically. In some embodiments, the presentinvention pertains to analytes that are target nucleic acid sequencesthat comprise or are in a “target nucleic acid” or a “targetpolynucleotide” or a “target oligonucleotide.” A composition, kit, ormethod of the invention can be used for an “analyte-specific reagent” todetect an analyte comprising a target nucleic acid sequence in a sample.

[0321] With respect to the present invention, an analyte is oftenassociated with a biological entity that is present in a sample if andonly if the analyte is present. Such biological entities include viroids(analyte is, e.g., a segment of a viroid nucleic acid sequence); viruses(analyte is, e.g., a sequence in the viral genome); other microorganisms(analyte is, e.g., a sequence in the genome or the RNA of themicroorganism); abnormal cells, such as cancer cells (analyte is, e.g.,a sequence in an oncogene); or an abnormal gene (analyte is, e.g., asequence in a gene segment that includes the altered bases which renderthe gene abnormal or in a messenger RNA segment that includes alteredbases as a result of having been transcribed from the abnormal gene).

[0322] However, in some embodiments of the invention an analyte can bechemically synthesized sequence or derived in the laboratory for aparticular purpose, rather than from a natural source. By way ofexample, but not of limitation, the analyte can be a chemicallysynthesized oligonucleotide tag that comprises a target sequence that iscovalently or non-covalently attached to an analyte-binding substancesuch as an antibody in order to indirectly detect another analyte in thesample which is bound by the analyte-binding substance. Alternatively,as discussed in greater detail later in the specification, theoligonucleotide tag that is attached or joined to an analyte-bindingsubstance can be referred to as a “target sequence” or a “targetsequence tag,” even though it is used to detect and/or quantify aprotein, lipid, carbohydrate or another analyte by detecting theanalyte-binding substance to which the target sequence is joined.

[0323] From the description of analytes, it is apparent that the presentinvention has widespread applicability, including in applications inwhich nucleic acid probe hybridization assays or immunoassays are oftenemployed. Thus, among other applications, the invention is useful indiagnosing diseases in plants and animals, including humans; and intesting products, such as food, blood, and tissue cultures, forcontaminants.

[0324] A “target” of the present invention is a biological organism ormaterial that is the reason or basis for which a diagnostic assay isperformed. By way of example, but not of limitation, an assay of thepresent invention may be performed to detect a target that is a viruswhich is indicative of a present disease or a risk of future disease(e.g., HIV which is believed to result in AIDS), or a target that is agene which is indicative of antibiotic resistance (e.g., an antibioticresistance gene in an infectious pathogenic bacterium), or a target thatis a gene which, if absent, may be indicative of disease (e.g., adeletion in an essential gene). In developing assays according to thepresent invention, it is important to identify target analytes thatyield assay results that are sufficiently specific, accurate, andsensitive to be meaningful related to the presence or condition of thetarget. A target analyte that is a sequence in a “target polynucleotide”or a “target nucleic acid” comprises at least one nucleic acid moleculeor portion of at least one nucleic acid molecule, whether said moleculeor molecules is or are DNA, RNA, or both DNA and RNA, and wherein eachsaid molecule has, at least in part, a defined nucleotide sequence. Thetarget polynucleotide may also have at least partial complementaritywith other molecules which can be used in an assay, such as, but notlimited to, capture probes. By way of example, in one embodiment, acapture probe for this purpose is complementary to a different region ofa target nucleic acid than the target sequence and may have a moiety,such as a biotin moiety, that permits immobilization of the targetnucleic acid on a surface, such as a surface to which streptavidin isattached.

[0325] The target polynucleotide may be single- or double-stranded. Atarget sequence of the present invention may be of any length. However,it must comprise a sequence of sufficient sequence specificity andlength so as to be useful for its intended purpose. By way of example,but not of limitation, a target sequence that is to be detected usingtarget sequence-complementary target probes must have a sequence ofsufficient sequence specificity and length so as remain hybridized bysaid target probes under assay hybridization conditions whereinsequences that are not target sequences are not hybridized. A targetsequence in a target polynucleotide having sufficient sequencespecificity and length for an assay of the present invention may beidentified, using methods known to those skilled in the art, bycomparison and analysis of nucleic acid sequences known for a target andfor other sequences which may be present in the sample. For example,sequences for nucleic acids of many viruses, bacteria, humans (e.g., forgenes and messenger RNA), and many other biological organisms can besearched using public or private databases, and sequence comparisons,folded structures, and hybridization melting temperatures (i.e.,T_(m)′s) may be obtained using computer software known to thoseknowledgeable in the art.

[0326] A method of the present invention can be carried out on nucleicacid from a variety of sources, including unpurified nucleic acids, ornucleic acids purified using any appropriate method in the art, such as,but not limited to, various “spin” columns, cationic membranes andfilters, or salt precipitation techniques, for which a wide variety ofproducts are commercially available (e.g., MasterPure™ DNA & RNAPurification Kits from Epicentre Technologies, Madison, Wis., USA).Methods of the present invention can also be carried out on nucleicacids isolated from viroids, viruses or cells of a specimen anddeposited onto solid supports as described by Gillespie and Spiegelman(J. Mol. Biol. 12: 829-842, 1965), including solid supports on dipsticksand the inside walls of microtiter plate wells. The method can also becarried out with nucleic acid isolated from specimens and deposited onsolid support by “dot” blotting (Kafatos, et al., Nucl. Acids Res., 7:1541-1552, 1979); White, and Bancroft, J. Biol. Chem., 257: 8569-8572,1982); Southern blotting (Southern, E., J. Mol. Biol., 98: 503-517,1975); “northern” blotting (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205, 1980); and electroblotting (Stellwag, and Dahlberg, Nucl.Acids Res., 8: 299-317, 1980). The method can also be carried out fornucleic acids spotted on membranes, on slides, or on chips as arrays ormicroarrays. Nucleic acid of specimens can also be assayed by the methodof the present invention applied to water phase hybridization (Britten,and Kohne, Science, 161: 527-540, 1968) and water/organic interphasehybridizations (Kohne, et al., Biochemistry, 16: 5329-5341, 1977).Water/organic interphase hybridizations have the advantage of proceedingwith very rapid kinetics but are not suitable when an organicphase-soluble linking moiety, such as biotin, is joined to the nucleicacid affinity molecule.

[0327] The methods of the present invention can also be carried out onamplification products obtained by amplification of a naturallyoccurring target nucleic acid, provided that the target sequence in thetarget nucleic acid is amplified by the method used only if the targetnucleic acid is present in the sample. Suitable amplification methodsinclude, but are not limited to, PCR, RT-PCR, NASBA, TMA, 3SR, LCR, LLA,SDA (e.g., Walker et al., Nucleic Acids Res. 20:1691-1696, 1992), RCA,Multiple Displacement Amplification (Molecular Staging), ICAN™ or UCAN™0(TAKARA), Loop-AMP (EIKEN), and SPIA™ or Ribo-SPIA™ (NuGENTechnologies). There are various reasons for using a nucleic acid thatis a product of another amplification method as a target nucleic acidfor an assay of the present invention, such as, but not limited to, forobtaining more sensitive detection of targets, greater specificity, orto decrease the time required to obtain an assay result.

[0328] The methods of the invention can also be carried out on nucleicacids isolated from specimens and deposited onto solid supports bydot-blotting, or by adsorption onto walls of microtiter plate wells orsolid support materials on dipsticks, on membranes, on slides, or onchips as arrays or microarrays. The amplified target-complementarysequences of target probes of the invention can also be hybridized tooligonucleotides or nucleic acids attached to or deposited on slides,chips or other surfaces, such as, but not limited to arrays ormicroarrays, for detection and identification.

[0329] Still further, the methods of the invention are applicable todetecting target sequences in cellular nucleic acids in whole cells froma specimen, such as a fixed or paraffin-embedded section, or frommicroorganisms immobilized on a solid support, such as replicα-platedbacteria or yeast. In some embodiments, the methods of the invention canbe used to amplify and/or detect target nucleic acid sequences in livingcells.

[0330] The invention is also not limited to detection of analytescomprising a target nucleic acid. The present invention provides assays,methods, compositions and kits for detection and quantification of ananalyte of any type in a sample.

[0331] C. Target Sequences Comprising Target Nucleic Acids in a Sampleor a Target Sequence Tag Joined to an Analyte-Binding Substance

[0332] The term “target nucleic acid sequence” or “target sequence”refers to the particular nucleotide sequence of the target nucleicacid(s) that is/are to be detected. A “target sequence” comprises one ormore sequences within one or more target nucleic acids, which targetnucleic acid can be naturally occurring in a sample or a target sequencetag that is joined or attached to an analyte-binding substance.

[0333] The target nucleic acid may be either single-stranded ordouble-stranded and may include other sequences besides the targetsequence. A target nucleic acid is sometimes referred to morespecifically by the type of nucleic acid. By way of example, but not oflimitation, a target nucleic acid can be a “target RNA” or an “RNAtarget,” or a “target mRNA,” or a “target DNA” or a “DNA target.”Similarly, the target sequence can be referred to as “a target RNAsequence” or an “RNA target sequence”, or as a “target mRNA sequence” ora “target DNA sequence,” or the like. In some embodiments, the targetsequence comprises one or more entire target nucleic acids. In otherembodiments, which are more common, the target sequence comprises only aportion of one or more nucleic acid molecules. The term “targetsequence” sometimes is also used to refer to the particulartarget-complementary nucleotide sequences that is/are present in thetarget-complementary “target probes” used in a method or assay of theinvention, but more preferably, these sequences are referred to as“target-complementary sequences.” The term “target sequence” refers onlyto that portion of the sequence of a target nucleic acid for which acomplementary sequence is present in a target probe of the invention. Insome embodiments of the invention, multiple target probes are used,including target probe sets that are complementary to other targetsequences in a target nucleic acid, which other target sequences can beon the same or the opposite nucleic acid strand of the same targetnucleic acid, or on another target nucleic acid in the sample or joinedto another analyte-binding substance. In some of those embodiments, atranscription promoter is present in a target probe that iscomplementary to only one strand of the target sequence, and in otherembodiments, a transcription promoter is present in two different targetprobes—one that is complementary to a target sequence on one strand, andthe other that is complementary to a complementary target sequence onthe other strand, wherein the transcription promoters can be the same ordifferent in each case. In most embodiments of the invention, the targetsequence in a method or assay of the invention will be a known sequenceor one of a small number of known sequences, such as, but not limited toone or a small number of sequences comprising known specific mutationsor single nucleotide polymorphisms (SNPs), or one of known sequencesthat are specific for and identify a particular organism or group ofclosely-related organisms. In some embodiments, wherein a method orassay of the invention is used to distinguish between two or more targetsequences that differ by a single nucleotide, we sometimes refer to thespecific nucleotide that differs between otherwise identical sequencesas a “target nucleotide.” A “target nucleotide” is part of a targetsequence and comprises the nucleotide position that differs between“wild-type” or “normal” alleles and single-base “mutant” alleles, or thenucleotide that differs between different “wild-type” alleles thatcomprise different single-nucleotide polymorphisms (SNPs) for aparticular nucleotide position in a target nucleic acid.

[0334] A target nucleic acid of the present invention comprising atarget sequence to be detected and/or quantified includes nucleic acidsfrom any source in purified or unpurified form. As discussed in greaterdetail herein above, target nucleic acids can be any DNA, including, butnot limited to, dsDNA and ssDNA, such as mitochondrial DNA, chloroplastDNA, chromosomes, plasmids or other episomes, the genomes of bacteria,yeasts, viruses, viroids, mycoplasma, molds, or other microorganisms, orgenomes of fungi, plants, animals, or humans, or target nucleic acidscan be any RNA, including, but not limited to, tRNA, mRNA, rRNA,mitochondrial RNA, chloroplast RNA, or target nucleic acids can bemixtures of DNA and RNA, including, but not limited to, mixtures of theabove nucleic acids or fragments thereof or DNA-RNA hybrids. The targetnucleic acid can be only a minor fraction of a complex mixture such as abiological sample and can be obtained from various biological materialsby procedures known in the art. As discussed herein above, methods forpurification of a target nucleic, if further purification is necessary,are also known in the art.

[0335] An initial step prior to amplification of a target nucleic acidsequence is rendering the target nucleic acid single-stranded. If thetarget nucleic acid is a double-stranded DNA (dsDNA), the initial stepis target denaturation. The denaturation step may be thermaldenaturation or any other method known in the art, such as alkalitreatment. Thus, in some embodiments of the invention in which thetarget nucleic acid in a sample is DNA, the ssDNA target sequencecomprises either ssDNA that is present in a biological sample or ssDNAthat is obtained by denaturation of dsDNA in the sample.

[0336] In other embodiments, the ssDNA target sequence comprises ssDNAthat is obtained as a result of a “primer extension reaction,” meaningan in vitro or in vivo DNA polymerization reaction using either ssDNA ordenatured dsDNA that is present in the sample as a template and anoligonucleotide as a primer under DNA polymerization reactionconditions. In some embodiments the target nucleic acid in the sample orthe primer extension product, or both, are made into smaller DNAfragments by methods known in the art in order to generate a DNA targetsequence for use in the methods of the invention.

[0337] If a target nucleic acid is RNA, the initial step may be thesynthesis of a single-stranded cDNA. Techniques for the synthesis ofcDNA from RNA are known in the art. Thus, in some embodiments of theinvention, which are preferred embodiments, the ssDNA target sequencecomprises first-strand cDNA obtained by reverse transcription of the RNAtarget, meaning an in vitro reaction that utilizes an RNA present in asample as a template and a nucleic acid oligonucleotide that iscomplementary to at least a portion of a sequence of the RNA template asa primer in order to synthesize ssDNA using an RNA-dependent DNApolymerase (i.e., reverse transcriptase) under reaction conditions. Insome embodiments, a first-strand cDNA for use in methods of theinvention is synthesized in situ in cells or tissue in a tissue sectionusing methods such as those described in U.S. Pat. Nos. 5,168,038;5,021,335; and 5,514,545, which are incorporated herein by reference.

[0338] D. Target Probes of the Invention; Simple Target Probes; PromoterTarget Probes; Signal Target Probes; Monpartite Target Probes; BipartiteTarget Probes

[0339] A “target probe” of the present invention is a linearsingle-stranded oligonucleotide that comprises at least one sequencethat is “a target-complementary sequence,” meaning a sequence that iscomplementary to a portion of a target sequence comprising a targetnucleic acid or a target sequence tag, and wherein the target probe isused in an assay or method of the invention.

[0340] The size and composition of a target-complementary sequence canvary. However, the target-complementary sequences of all target probesof the invention must be of sufficient length and nucleotide compositionso as to anneal with specificity to a complementary target sequence withwhich it is perfectly based-paired under the conditions used in theassay or method for annealing of target probes to the target sequenceand for ligation of the target probes that are annealed to the targetsequence, under which conditions, target probes that are notcomplementary to the target sequence do not remain annealed and, if notperfectly basepaired at the ligation junction, do not ligate. In orderto meet these conditions, those with knowledge in the art willunderstand that the length of a target-complementary sequence can varybased in part on its sequence and on the T_(m) of that sequence, and onthe temperature and other reaction conditions that are used forannealing of the target probes and ligation of the target probes on thetarget sequence. In general, a target-complementary sequence of a targetprobe will comprise at least four nucleotides if thetarget-complementary sequences are annealed to the target sequence andligated at a temperature that is less than or equal to about 30 degreesC, or at least about eight nucleotides if the target-complementarysequences are annealed to the target sequence and ligated at atemperature that is greater than about 30 degrees C. Preferably, atarget-complementary sequence of a target probe that is complementary tothe 5′-end or to the 3′-end of the target sequence comprises about 10 toabout 100 nucleotides, and most preferably, about 10 to about 50nucleotides. However, based on this description of the invention, thosewith knowledge in the art will know how to empirically determine theoptimal lengths of target-complementary sequences for target probes forparticular target sequences, and under the particular conditions, whichconditions can vary with respect to factors such as but not limited totemperature, ionic strength, concentration of co-solvents such as butnot limited to betaine, or other factors.

[0341] Further, the length of one target-complementary sequence of abipartite target probe can be different than the other. Also, the lengthof a target-complementary sequence of one monopartite target probe canbe different than the lengths of target-complementary sequences of othermonopartite target probes used in the assay or method.

[0342] In general, the sequence of a target probe that is complementaryto the 3′-end of a target sequence is designed to be longer than thesequence of a target probe that is joined to a sense promoter sequenceand that is complementary to the 5′-end of the target sequence, althoughthe sequence of a target probe that is complementary to the 3′-end ofthe target sequence need not be longer, and can be about the same sizeor even shorter than the sequence that is complementary to the 5′-end ofthe target sequence. However, by using a target probe with a longersequence that is complementary to the 3′-end of the target sequence, thehybridization complex between that target probe and the target sequencewill be more stable so that the ability to form a ligation junction willbe more dependent on the annealing of the target-complementary sequencethat is joined to the sense promoter sequence and that anneals to the5′-end of the target sequence.

[0343] It is preferable that the length of the target-complementarysequence that is joined to the sense promoter sequence comprises asufficient number of nucleotides so as anneal to the 5′-end of thetarget sequence with specificity under the conditions of the assay ormethod, but is optimized so that transcription of saidtarget-complementary sequence is minimized unless and until it isligated to another target-complementary sequence that is adjacentlyannealed on the target sequence. Without being bound by theory, itappears that the optimal length of the target-complementary sequencethat is joined to the sense promoter sequence can vary for different RNApolymerases. N4 mini-vRNAP, unlike T7 RNAP, does not have RNA:DNA hybridunwinding activity and EcoSSB Protein appears to be responsible fordisplacing the RNA transcript from the template strand. The amount ofEcoSSB-activated displacement appears to vary with the total length ofthe template strand and the length of the transcript (Davidova, E. andRothman-Denes, Proc. Natl. Acad. Sci. USA, 100: 9250-9255, 2003).Therefore, if a target probe comprises an N4 vRNAP promoter, the lengthof the target-complementary sequence that is joined to the N4 promotersequence is designed, based on the information of Davidova et al., sothat a transcript made by an N4 mini-vRNAP will either not be displacedor at least, EcoSSB-activated displacement is minimized from thetarget-complementary sequence unless and until this sequence is ligatedto an adjacent target-complementary sequence that is annealed to thetarget sequence to make a transcription substrate of the invention. Thisdiffers from RNA polymerases, such as T7-like RNAPs, which have RNA:DNAhybrid unwinding activity and displace both long and very shorttranscripts from the template. Thus, target probes can be designed forN4 mini-vRNAP enzymes so that background transcription from an unligatedN4 promoter-containing target probe is less than from an unligated T7sense promoter-containing target probe under transcription conditions inthe presence of the respective cognate RNA polymerase.

[0344] If there is a gap between target-complementary sequences of abipartite target probe or between target-complementary sequences of apromoter target probe and another monopartite target probe when annealedto the 5′-end and the 3′-end, respectively, of the target sequence, andone or more simple target probes is used in the assay or method to fillthe gap, then the simple target probe(s) that is/are used to fill thegap can be of any length so long as they provide suitable ligationjunctions for joining to the target-complementary sequences that areannealed to the 3′- and 5′-ends of the target sequence.

[0345] In general, target probes comprise deoxyribonucleotides havingcanonical nucleic acid bases and internucleoside linkages, althoughmodified sugars, bases or internucleoside linkages can be used for aparticular purpose as discussed elsewhere herein.

[0346] One type of target probe of the invention, which is called a“simple target probe,” comprises a linear single-strandedoligonucleotide comprising only a sequence that is complementary to onecontinuous portion of a target sequence.

[0347] Another embodiment of a target probe of the invention is a“promoter target probe.” A “promoter target probe” comprises a5′-portion and a 3′-portion, wherein, said 5′-portion comprises asequence that is complementary to the most 5′-portion of a targetsequence, and said 3′-portion comprises a sequence that serves as afunctional transcription promoter for an RNA polymerase that synthesizesRNA under transcription conditions using said single-strandedtranscription promoter and ssDNA that is 5′- of said promoter (withrespect to the same strand) as a template. Optionally, a promoter targetprobe can also comprise other “optional sequences” that do not comprisea target-complementary sequence or a promoter sequence, which optionalsequences, if present, are 3′- of said promoter sequence in saidpromoter target probe. Such optional sequences in said promoter targetprobe can serve other functions in a method or assay of the invention.

[0348] In embodiments of the invention that result in lineartranscription substrates rather than circular transcription substrates,as discussed below, another embodiment of a target probe of theinvention that can be used, but which is optional, is a “signal targetprobe.” A “signal target probe” comprises a 3′-portion and a 5′-portion,wherein, the 3′-end portion of said signal probe comprises a sequencethat is complementary to the most 3′-portion of a target sequence, andsaid 5′-portion comprises a “signal sequence,” wherein said signalsequence comprises a sequence that is detectable in some way, directlyor indirectly, following transcription of said signal sequence that isjoined, in the presence of a target sequence, to a target-complementarysequence and a single-stranded promoter sequence during an assay ormethod of the present invention. In vitro transcription of the signalsequence results in synthesis of RNA that is complementary to saidsignal sequence, which in turn is detectable in some way (depending onwhat the signal sequence encodes) in an assay or method of theinvention. A signal target probe can also comprise other “optionalsequences” that do not comprise the signal sequence, which sequences canserve another function in a method or assay of the invention, or theycan have no function, other than to connect the signal sequence to oneor more other sequences.

[0349] “Monopartite target probes” of the present invention are targetprobes that comprise only one sequence that is complementary to oneportion of a target sequence. The target-complementary sequence in amonopartite target probe is not interrupted by any other sequence thatis not complementary to the target sequence. Promoter target probes andsignal target probes are monopartite target probes that are used togenerate linear transcription substrates in some embodiments of assaysand methods of the invention. Simple target probes are monopartitetarget probes that can be used in embodiments of the invention togenerate either linear transcription substrates or circulartranscription substrates, as discussed herein below. Simple targetprobes are monopartite target probes that are used in embodiments of theinvention in order to fill at least a portion of a gap region betweentarget-complementary sequences of other target probes that are notcontiguous when annealed to a target sequence. For example, one or moresimple target probes can be used in methods and assays of the inventionthat generate a linear transcription substrate by annealing to a targetsequence between the sequences of the target sequence to which thetarget-complementary sequences of a promoter target probe and a signaltarget probe anneal. FIG. 18 illustrates monopartite target probes andshows one embodiment of how monopartite target probes are oriented whenannealed to a target sequence. In still other embodiments of theinvention, the target-complementary sequences of the promoter targetprobe and the signal target probe are contiguous or adjacent when theyare annealed to a target sequence and a simple target probe is not used.In still another embodiment, the target-complementary sequences of thepromoter target probe and the signal target probe are not contiguouswhen they are annealed to a target sequence, but rather than using asimple target probe to anneal to the gap region on the target sequencebetween the the target-complementary sequences of the promoter targetprobe and the signal target probe, the gap is “filled” by DNA polymeraseextension from the 3′-end of the signal target probe.

[0350] One or more simple target probes can also be used in embodimentsof methods and assays of the invention that generate a circulartranscription substrate, in which case, the simple target probe(s)anneal to a target sequence between the regions of the target sequenceto which the target-complementary sequences at the ends of a bipartitetarget probe anneal.

[0351] Thus, other embodiments of the invention, which are preferredembodiments, use a bipartite target probe and generate a circulartranscription substrate. A “bipartite target probe” is referred to as“bipartite” because it comprises two different target-complementarysequences, each of which is complementary to a different portion of atarget sequence, which target-complementary sequences are separatedwithin the bipartite target probe by other sequences that are notcomplementary to the target sequence. Thus, the target-complementarysequences in a bipartite target probe are in two parts or “bipartite.” Abipartite target probe comprises a ssDNA that has a 5′-end thatresembles a promoter target probe and a 3′-end that resembles either asimple target probe or a signal target probe. Thus, the 5′-end of abipartite target probe has a sequence that is complementary to the most5′-portion of a target sequence and, then on the same strand, 3′- of thetarget-complementary sequence, a functional transcription promoter foran RNA polymerase that can synthesize RNA under transcription conditionsusing said single-stranded transcription promoter and ssDNA that iscovalently joined 5′- of said promoter as a template. The 3′-end of abipartite target probe comprises a sequence that is complementary to themost 3′-end portion of said target sequence. If it is used, a signalsequence can be 5′- of the target-complementary sequence at the 3′-endof a bipartite target probe, although a signal sequence does not need tobe contiguous with or immediately adjacent to the target-complementarysequence at the 3′-end of a bipartite target probe. Other sequences thatcan have other functions or that have no function other than to join thetwo sequences can be between a target-complementary sequence at the3′-end and a signal sequence of a bipartite target probe. Thetarget-complementary sequences of a bipartite target probe need not becontiguous or immediately adjacent when they are annealed to a targetsequence. If the bipartite target-complementary sequences are notcontiguous or immediately adjacent when they are annealed to a targetsequence, then one or more simple target probes that are complementaryto the portions of the target sequence between the target-complementarysequences of the bipartite target probe can be used in some embodimentsof methods or assays of the invention.

[0352] Alternatively, in other embodiments of the invention in which thebipartite target-complementary sequences are not contiguous orimmediately adjacent when they are annealed to a target sequence, a DNApolymerase can be used to “fill in” where there is no target probeannealed to the target sequence by primer-extending from the 3′-hydroxylend of a bipartite target probe that is annealed to a target sequenceusing the target sequence as a template.

[0353]FIG. 19 illustrates a bipartite target probe of the invention andshows how different sequence portions of a bipartite target probe areoriented with respect to each other when said bipartite target probe isfree in solution and when it is annealed to a target sequence. Theembodiment illustrated in FIG. 19 shows a bipartite target probecomprising target-complementary sequences at each end that arecontiguous or adjacent when annealed to a target sequence. As discussedabove, the invention also comprises other embodiments of bipartitetarget probes wherein the target-complementary sequences at each endthat are not contiguous or adjacent when annealed to a target sequence.In those embodiments, the “gap” between the target-complementarysequences of the bipartite target probe can be “filled” using one ormore simple target probes or by DNA polymerase extension from the 3′-endof the bipartite target probe using the target sequence as a template.

[0354] In general, all target probes of the invention, including bothmonopartite and bipartite target probes that are joined with a ligase ina method or assay of the invention, will have a phosphate group at their5′-end and a hydroxyl group at their 3′-end. The 5′-ends of targetprobes that are not joined with a ligase to the 3′-end of another targetprobe in a method or assay of the invention do not have a phosphategroup on their 5′-ends. The only exceptions will be in those embodimentsthat use another joining method, such as, but not limited to a chemicaljoining method or a topoisomerase-mediated joining method.

[0355] In some embodiments of the invention, such as, but not limited tothe embodiment illustrated in FIG. 26, secondary or additionalamplification of a target sequence and/or a signal sequence is obtainedby using a “second target probe,” which second target probe can compriseeither: (i) “second monopartite target probes” comprising a “secondpromoter target probe” and either a “second signal target probe,” if asignal sequence is present, or a “second simple target probe,” and oneor more additional “second simple target probes; or (ii) a “secondbipartite target probe.” If a second target probe is used, then thetarget probes that are complementary to the target sequence are referredto as “first target probes.” A second target probe is generallyidentical to a first target probe except with respect to thetarget-complementary sequence of said target probe. Thus, the sequenceat the 5′-end of a second promoter target probe or at the 5′-end of asecond bipartite target probe, rather than being complementary to atarget sequence, is complementary to the target-complementary sequenceat the 3′-end of the first signal target probe or to thetarget-complementary sequence at the 3′-end of the bipartite targetprobe, respectively. Similarly, the sequence at the 3′-end of a secondsignal target probe or at the 3′-end of a second bipartite target probe,rather than being complementary to a target sequence, is complementaryto the target-complementary sequence at the 5′-end of the first promotertarget probe or to the target-complementary sequence at the 5′-end ofthe bipartite target probe, respectively. A second simple target probe,rather than being complementary to a target sequence, is complementaryto a first target probe. A second target probe can also be referred toas an “target amplification probe,” which can comprise either: (i)“monopartite target amplification probes” comprising a “promoter targetamplification probe” and either a “signal target amplification probe,”if a signal sequence is present, or a “simple target amplificationprobe,” and one or more additional “simple target amplification probes;or (ii) a “bipartite target amplification probe.”

[0356] E. Design of Target Probes of the Invention for Detection ofMutations, Including Single Nucleotide Polymorphisms (SNP's)

[0357] In embodiments of a method or assay of the invention todistinguish between two or more target sequences that differ by a singlenucleotide, wherein the specific nucleotide that differs betweenotherwise identical sequences is referred to as a “target nucleotide,”the target probes used in said assay or method are designed in order tobe able to distinguish said target nucleotide(s). In preferredembodiments of assays and methods to detect a single-nucleotidedifference in a target sequence, the nucleotide of a target probe of theinvention that is complementary to the target nucleotide compriseseither the 5′-end of a promoter target probe if the assay or methodgenerates a linear transcription substrate, or the nucleotide at the5′-end of a bipartite target probe if the assay or method generates acirular transcription substrate. Then, if a target nucleotide is presentin a target sequence in a sample, the complementary nucleotide at the5′-end of the respective promoter target probe or the bipartite targetprobe will anneal thereto and will be ligated, respectively, either tothe 3′-end of an adjacently-annealed monopartite target probe or to anadjacently-annealed 3′-end of the bipartite target probe. If the 5′-endof the promoter target probe or the 5′-end of the bipartite target probeis not complementary to the target nucleotide, it will not annealthereto, and said 5′-end will not be ligated to the 3′-end of anadjacently-annealed monopartite target probe or the 3′-end of thebipartite target probe, respectively, during the ligation process. Thatis, the non-complementarity of the 5′-end of a target probe with atarget nucleotide in a target sequence, when the target probes of anassay or method are annealed to the target sequence prevents ligation ofsaid 5′-end with a 3′-hydroxyl end, so that a transcription substrate isnot formed. Although the preferred nucleotide of a target probe of theinvention that is complementary to the target nucleotide compriseseither the 5′-end of a promoter target probe if the assay or methodgenerates a linear transcription substrate, or the nucleotide at the5′-end of a bipartite target probe if the assay or method generates acirular transcription substrate, the invention also comprises otherembodiments of target probes in which the nucleotide that iscomplementary to the target nucleotide comprises a different nucleotidein a monopartite or bipartite target probe. Thus, in embodiments ofassays or methods using monopartite target probes in which there is no“gap” between the sites on the target sequence to which a promotertarget probe and a signal target probe if a signal sequence is present,or a simple target probe if a signal sequence is not present, then thenucleotide that is complementary to the target nucleotide can compriseeither the 3′-end of the signal target probe if a signal sequence ispresent, or the 3′-end of the simple target probe if a signal sequenceis not used. Similarly, in embodiments of assays or methods using abipartite target probe in which there is no “gap” between the sites onthe target sequence to which the 5′-end and the 3′-end of said bipartitetarget probe anneal, then the nucleotide that is complementary to thetarget nucleotide can comprise the 3′-end of said bipartite targetprobe. In embodiments of assays or methods using monopartite targetprobes in which there is a gap between the sites on the target sequenceto which a promoter target probe and a signal target probe if a signalsequence is present, or a simple target probe if a signal sequence isnot present, wherein one or more simple target probes are used to “fillthe gap,” then the nucleotide that is complementary to the targetnucleotide can comprise a nucleotide at either the 3′-end or the 5′-endof one of said simple target probes that is used to fill the gap.Preferably, the nucleotide that is complementary to the targetnucleotide comprises a nucleotide at either the 3′-end or the 5′-end ofa simple target probe used to fill the gap that anneals to the targetsequence adjacent to the promoter target probe, and most preferably, thenucleotide that is complementary to the target nucleotide comprises anucleotide at the 3′-end of said simple target probe. In embodiments ofassays or methods using bipartite target probes in which there is a gapbetween the sites on the target sequence to which the ends of thebipartite target probe anneal, wherein one or more simple target probesare used to fill the gap, then the nucleotide that is complementary tothe target nucleotide can comprise a nucleotide at either the 3′-end orthe 5′-end of one of said simple target probes that is used to fill thegap. Preferably, the nucleotide that is complementary to the targetnucleotide comprises a nucleotide at either the 3′-end or the 5′-end ofa simple target probes used to fill the gap that anneals to the targetsequence adjacent to the 5′-end of said bipartite target probe, and mostpreferably, the nucleotide that is complementary to the targetnucleotide comprises a nucleotide at the 3′-end of said simple targetprobe. It will be understood by those with knowledge in the art that oneor more 5′-terminal or 3′-terminal nucleotide positions of a targetprobe used in an assay or method to detect a particular targetnucleotide in a target sequence may not comprise a sequence that willanneal with specificity to said target sequence, such as, when saidtarget nucleotide is part of a target sequence that has a low T_(m) withrespect to said target-complementary sequence of said target probe. Insuch cases, those with knowledge in the art will know how to evaluateand, without undue experimentation, how to choose which of thosepossible 5′-terminal and 3′-terminal nucleotide positions of all of thetarget probes used in said method or assay comprises the “bestnucleotide” to be complementary to said target nucleotide, wherein saidbest nucleotide results in the greatest specificity and sensitivity insaid assay or method.

[0358] F. Signal Sequences in Signal Target Probes or Bipartite TargetProbes of the Invention

[0359] A method or assay of the invention does not need to use a signalsequence. The use of a signal sequence or a signal target probe in amethod or assay of the invention is optional. If a signal sequence isused in an embodiment, the invention is not limited with respect toparticular signal sequences that can be used in signal target probes orbipartite target probes of the invention. A signal sequence can compriseany sequence that generates a detectable signal or that enablessensitive and specific detection, whether directly or indirectly, of thegeneration of an RNA transcription product encoded by the signalsequence. Preferably, a signal sequence encodes an RNA product thatresults in additional amplification or more sensitive detection.

[0360] By way of example, but not of limitation, one signal sequencethat can be used in a signal target probe or a bipartite target probe ofthe present invention is a sequence that encodes a substrate for areplicase, such as, but not limited to, Q-beta replicase or a partial orinterrupted sequence for a substrate for a replicase, such as, but notlimited to, Q-beta replicase. Q-beta replicase substrates and methodsthat can be used for making and using signal sequences that encode apartial or interrupted replicase substrate for signal target probes aredescribed in U.S. Pat. No. 6,562,575, incorporated herein by reference.A complete sequence for a replicase substrate is preferred in a signalprobe of the present invention, but a sequence of a partial orinterrupted replicase substrate is used in embodiments that requirereduced background signal (or “noise”) and greater sensitivity. If thetime for appearance of a signal in an assay or method is shortened byamplifying the amount of transcription product using other methodsdescribed herein, it is less likely that a sequence for a partial orinterrupted replicase substrate, rather than for a complete replicasesubstrate, is needed to obtain a good signal to noise ratio in the assayor method. Once an RNA that is a substrate for Q-beta replicase issynthesized, incubation of said RNA substrate with Q-beta replicaseresults in replication of the substrate, thereby resulting in additionalamplification of the signal and more sensitive, though indirect,detection of the presence of a target sequence.

[0361] A “replicase” according to the invention is an enzyme thatcatalyzes exponential synthesis (i.e., “replication”) of an RNAsubstrate. The replicase can be from any source for which a suitableexponentially replicatable substrate can be obtained for use in theinvention. Preferably, the replicase is an RNA-directed RNA polymerase.In preferred embodiments, the replicase is a bacteriophage replicase,such as Q-beta replicase, MS2 replicase, or SP replicase. In the mostpreferred embodiment, the replicase is Q-beta replicase. In otherpreferred embodiments, the replicase is isolated from eucaryotic cellsinfected with a virus, such as, but not limited to, cells infected withbrome mosaic virus, cowpea mosaic virus, cucumber mosaic virus, or poliovirus. In another embodiment, the replicase is a DNA-directed RNApolymerase, in which case, a T7-like RNA polymerase (as defined in U.S.Pat. No. 4,952,496) is preferred, and T7 RNA polymerase (Konarska, M.M., and Sharp, P. A., Cell, 63: 609-618, 1990) is most preferred. Thereplicase can be prepared from cells containing a virus or from cellsexpressing a gene from a bacteriophage or a eukaryotic virus cloned intoa plasmid or other vector.

[0362] If Q-beta replicase is used, replication of a Q-beta replicasesubstrate can be carried out substantially according to the protocol ofKramer et al. (J. Mol. Biol., 89: 719-736, 1974). Briefly, an RNAsubstrate is incubated at 37° C. in a reaction mixture containing about20-50 micrograms of Q-beta replicase per ml, 40-100 mM Tris-HCl (pH7-8), about 10-12 mM MgCl₂, and about 200-400 micromolar each of ATP,CTP, UTP and GTP. If desired, one of the NTPs can be labeled with afluorescent or other dye, or the replication products can be detectedusing another method, such as but not limited to by detection offluorescence that results from intercalation of a dye such as ethidiumbromide.

[0363] In embodiments that use Q-beta replicase, it is preferred thatthe sequence of the recombinant substrate or template be derived fromthe sequence of an RNA in the following group: midivariant RNA (MDV-1RNA), microvariant RNA, nanovariant RNA, CT RNA, RQ135 RNA, RQ120 RNA,and other variants or Q-beta RNA, which are known in the art. Once asubstrate for a replicase is identified, improved substrates can beobtained, if desired, by serial transfer and selection of higheryielding products from successive reactions, including prolongedreactions. Further, improved substrates can be obtained by random orsite-directed modification of a known substrate, followed by serialtransfer and selection to select new substrates that result in greaterincorporation of UTP during replication.

[0364] Another signal sequence that can be used is an expressable genefor an enzyme that has a substrate that results in a colored orfluorescent or otherwise detectable product. By way of example, but notof limitation, a gene for a green fluorescent protein (GFP) can be used.In that case, in vitro transcription of a transcription substrategenerated by target-dependent annealing and ligation of target probesresults in an RNA transcript that encodes a GFP. In the presence of anin vitro translation system, a detectable GFP is synthesized. There aremany other genes that encode enzymes that can be used to generatedetectable signals following coupled or stepwise in vitro transcriptionand translation. By way of example, but not of limitation, signalsequences comprising genes for phosphatases or beta-galactosidases canbe used, together with a suitable substrate that generates a colored,fluorescent or chemiluminescent product. A large number of enzymes andcoenzymes, as well as enzyme combinations that are useful in a signalproducing system are indicated In U.S. Pat. No. 4,275,149 and U.S. Pat.No. 4,318,980, which disclosures are incorporated herein in theirentirety by reference. Still further, a signal sequence can comprise abinding site for another molecule, such as, but not limited to, amolecular beacon that results in a signal. Those with knowledge in theart will know many other ways to design a signal sequence for use intarget probes of the invention, all of which are part of the presentinvention.

[0365] G. Other Optional Sequences in Target Probes of the Invention

[0366] A monopartite or a bipartite target probe of the presentinvention can optionally comprise other “optional sequences.” Optionalsequences, if present, can be 5′- of the target-complementary sequenceat the 3′-end and 3′- of the promoter sequence in the 5′-portion of abipartite target probe. Optional sequences, if present in a monopartitetarget probe, can be 5′- of the target-complementary sequence in asignal target probe or 3′- of the promoter sequence in a promoter targetprobe. By way of example, but not of limitation, other optionalsequences can comprise one or more transcription termination sequences,one or more capture sequence sites, one or more detection sequencesites, one or more address tag sites, one or more priming sites, one ormore sequences for another specific purpose, or one or more interveningsequences that have no function other than to link one portion of atarget probe to another portion. A capture sequence site can be a sitethat is complementary to another oligonucleotide, such as, but notlimited to an oligo with a biotin group, that facilitates capture of atarget sequence to a surface, such as a surface to which streptavidin isbound. A detection sequence site can be a sequence that is complementaryto an oligo used for detection, such as, but not limited to, a molecularbeacon. An address tag can be a sequence that is complementary to anoligonucleotide or a polynucleotide that is attached to a surface, suchas, but not limited to, a dipstick or a spot on an array or microarray.A priming site can be for a sequence that is complementary to anoligonucleotide primer, such as, but not limited to a primer for use inreverse transcription of an RNA transcript product of an assay or methodof the invention. These optional sequences can be of any length thatpermits stable and specific hybridization for the intended purpose andthat does not hinder the performance of an assay or method of theinvention.

[0367] H. Transcription Substrates of the Invention: CircularTranscription Substrates and Linear Transcription Substrates

[0368] As used herein, a “transcription substrate” according to thepresent invention comprises a ssDNA comprising: (i) a sequence thatserves as a functional transcription promoter for an RNA polymerase thatlacks helicase-like activity and that can use said promoter to initiatetranscription of a ssDNA sequence that is 5′- of said single-strandedpromoter in the same strand under transcription conditions; and (ii) asequence that comprises a contiguous target-complementary sequence thatresults from covalent joining in the presence of a target sequence ofeither: (a) at least two portions of a target-complementary sequencecomprising at least two monopartite target probes; or (b) at least twotarget-complementary portions of a sequence comprising a bipartitetarget probe; and wherein said transcription promoter is 3′- of saidcontiguous target-complementary sequence to which it is joined.Optionally, a transcription substrate of the invention can also haveadditional nucleic acid sequences, such as, but not limited to,detectable “signal sequences,” that are in the same DNA strand and 5′-of said contiguous target-complementary sequence, which in turn is inthe same DNA strand and 5′- of said transcription promoter sequence,wherein, in the absence of a transcription terminator sequence, bothsaid contiguous target-complementary sequence, and said additionalnucleic acid sequences, if present in said transcription substrate, aretranscribed by said RNA polymerase that recognizes said transcriptionpromoter under transcription conditions. However, a transcriptionsubstrate of the invention is not required to have said additionalnucleic acid sequences.

[0369] A transcription substrate typically has a transcriptioninitiation site at the 5′-end of the promoter sequence. A transcriptionsubstrate of the invention can also have one or more other sequencesthat are 5′- of the target-complementary sequence. By way of example,but not of limitation, a transcription substrate can have one or moretranscription termination sequences, one or more sites for DNA cleavageto permit controlled linearization of a circular transcriptionsubstrate, and/or other sequences or genetic elements for a particularpurpose, including, but not limited to, sequences that are transcribedby the RNA polymerase so as to provide additional regions ofcomplementarity in the RNA transcription products: (i) for annealing ofprimers for reverse transcription in order to make cDNA for additionalrounds of amplification; or (ii) for annealing of additional targetprobes for generation of additional transcription substrates by means ofadditional joining reactions using the RNA transcription product as aligation template (e..g., by using a different joining enzyme or joiningmethod on the RNA ligation template than the joining enzyme or joiningmethod that was used in the initial joining reaction of target probes onthe target sequence).

[0370] I. Hybridization or Annealing Processes of the Invention

[0371] “Hybridization” or “annealing” refers to the “binding” or“pairing” of complementary nucleic acid bases in one single-strandednucleic acid, peptide nucleic acid (PNA), or linked nucleic acid-PNAmolecule with another single-stranded nucleic acid, PNA, or linkednucleic acid-PNA molecule under “binding” or “annealing” or“hybridization” conditions.” The ability of two polymers of nucleic acidand/or PNA containing complementary sequences to find each other andanneal through base pairing interaction is a well-recognized phenomenon.The initial observations of the “hybridization” process by Marmur andLane (Proc. Nat. Acad. Sci. USA, 46: 453, 1960) and Doty, et al. (Proc.Nat. Acad. Sci. USA, 46: 461, 1960) have been followed by the refinementof this process into an essential tool of modern biology. Hybridizationoccurs according to base pairing rules (e.g., adenine pairs with thymineor uracil and guanine pairs with cytosine). Those with skill in the artwill be able to develop and make conditions which comprise bindingconditions or hybridization conditions for a particular target nucleicacid analytes or target sequence tag joined to a non-nucleic acidanalyte and target probes of an assay or method of the invention. Indeveloping and making binding conditions for particular target nucleicacid analytes or target sequence tags joined to non-nucleic acidanalytes with target probes an assay of the invention, as well as indeveloping and making hybridization conditions for otheroligonucleotides or polynucleotides which can be used, such as, but notlimited to capture probes, or detection probes such as molecularbeacons, certain additives can be added in the hybridization solution.By way of example, but not of limitation, dextran sulfate orpolyethylene glycol can be added to accelerate the rate of hybridization(e.g., Chapter 9, Sambrook, et al., Molecular Cloning: A LaboratoryManual, 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989), orbetaine can be added to the hybridization solution to eliminate thedependence of T_(m). on basepair composition (Rees, W. A., et al.,Biochemistry, 32, 137-144, 1993). However, other hybridizationconditions that do not use such additives can also be used in an assayor method of the invention.

[0372] The terms “degree of homology” or “degree of complementarity”refer to the extent or frequency at which the nucleic acid bases on onestrand (e.g., of the affinity molecule) are “complementary with” or“able to pair” with the nucleic acid bases on the other strand (e.g.,the analyte). Complementarity may be “partial,” meaning only some of thenucleic acid bases are matched according to base pairing rules, orcomplementarity may be “complete” or “total.” The length (i.e., thenumber of nucleic acid bases comprising the nucleic acid and/or PNAaffinity molecule and the nucleic acid analyte), and the degree of“homology” or “complementarity” between the affinity molecule and theanalyte have significant effects on the efficiency and strength ofbinding or hybridization when the nucleic acid bases on the affinitymolecule are maximally “bound” or “hybridized” to the nucleic acid baseson the analyte. The terms “melting temperature” or “T_(m)” are used asan indication of the degree of complementarity. The T_(m) is thetemperature at which a population of double-stranded nucleic acidmolecules becomes half dissociated into single strands under definedconditions. Based on the assumption that a nucleic acid molecule that isused in hybridization will be approximately completely homologous orcomplementary to a target polynucleotide, equations have been developedfor estimating the T_(m) for a given single-stranded sequence that ishybridized or “annealed” to a complementary sequence. For example, acommon equation used in the art for oligodeoxynucleotides is:T_(m)=81.5° C.+0.41(% G+C) when the nucleic acid is in an aqueoussolution containing 1 M NaCl (see e.g., Anderson and Young, QuantitativeFilter Hybridization, in Nucleic Acid Hybridization,1985). Other moresophisticated equations available for nucleic acids take nearestneighbor and other structural effects into account for calculation ofthe T_(m). Binding is generally stronger for PNA affinity molecules thanfor nucleic acid affinity molecules. For example the T_(m) of a 10-merhomothymidine PNA binding to its complementary 10-mer homoadenosine DNAis 73° C., whereas the T_(m) for the corresponding 10-mer homothymidineDNA to the same complementary 10-mer homoadenosine DNA is only 23° C.Equations for calculating the T_(m) for a nucleic acid are notappropriate for PNA. Preferably, a T_(m) that is calculated using anequation in the art, is checked empirically and the hybridization orbinding conditions are adjusted by empirically raising or lowering thestringency of hybridization as appropriate for a particular assay.

[0373] As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. With“high stringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “weak” or “low”stringency are often required when it is desired that nucleic acids thatare not completely complementary to one another be hybridized orannealed together.

[0374] With regard to complementarity, it is important for some assaysof the invention to determine whether the hybridization representscomplete or partial complementarity. For example, where it is desired todetect simply the presence or absence of pathogen DNA (such as from avirus, bacterium, fungi, mycoplasma, protozoan), it is only importantthat the hybridization method ensures hybridization when the relevantsequence is present. In those embodiments of the invention, conditionscan be selected where both partially complementary probes and completelycomplementary probes will hybridize.

[0375] However, the invention can also be used for assays to detectmutations, or genetic polymorphisms, or single nucleotide polymorphisms(SNPs). These embodiments of the invention require that thehybridization and other aspects of the method distinguish betweenpartial and complete complementarity. For example, human hemoglobin iscomposed, in part, of four polypeptide chains. Two of these chains areidentical chains of 141 amino acids (alpha chains) and two of thesechains are identical chains of 146 amino acids (beta chains). The geneencoding the beta chain is known to exhibit polymorphism. The normalallele encodes a beta chain having glutamic acid at the sixth position.The mutant allele encodes a beta chain having valine at the sixthposition. This difference in amino acids has a profound (most profoundwhen the individual is homozygous for the mutant allele) physiologicalimpact known clinically as sickle cell anemia. It is well known that thegenetic basis of the amino acid change involves a single base differencebetween the normal allele DNA sequence and the mutant allele DNAsequence. Thus, some embodiments of the invention are used for assaysthat can detect and distinguish even as small a difference as a singlebasepair in a target nucleic acid analyte.

[0376] J. Ligases and Ligation Processes of the Invention

[0377] In general, “ligation” refers to the joining of a5′-phosphorylated end of one nucleic acid molecule with the 3′-hydroxylend of another nucleic acid molecule by an enzyme called a “ligase,”although in some methods of the invention, the ligation can be effectedby another mechanism. With respect to ligation, a region, portion, orsequence that is “adjacent to” or “contiguous to” or “contiguous with”another sequence directly abuts that region, portion, or sequence.

[0378] The invention is not limited to a specific ligase. However,preferably the ligase is not active in ligating blunt ends and is highlyselective for ligation of a deoxyribonucleotide having a 5′-phosphateand a deoxyribonucleotide having 3′-hydroxyl group when these respective5′- and 3′-nucleotides are adjacent to each other when annealed to atarget sequence of a target nucleic acid. Ampligases Thermostable DNALigase Tth DNA ligase, and Tfl DNA Ligase (Epicentre Technologies,Madison, Wis., USA), or Tsc DNA Ligase (Prokaria Ltd., Reykjavik,Iceland) are NAD-dependent thermostable ligases that are not active onblunt ends and that ligate the 5′-phosphate and 3′-hydroxyl termini ofDNA ends that are adjacent to one another when annealed to acomplementary DNA molecule; these enzymes are preferred ligases inembodiments of the invention wherein a target sequence comprises DNA.Another DNA ligase that can be used in the methods of the invention fortarget sequences comprising DNA is Pfu DNA ligase as described by Mathuret al. (U.S. Pat. Nos. 5,700,672 and 6,280,998). Thermostable DNAligases are preferred in some embodiments because they can be cycledthrough multiple annealing-ligation-melting cycles, permitting multipletarget probe ligations for every target sequence present in a sample,and thus, increasing the sensitivity of the assay or method. However,the invention is not limited to the use of a particular ligase, or tothe use of a thermostable ligase and other suitable ligases thatfunction in the assays and methods of the invention can also be used.For example, T4 DNA ligase can be used in some embodiments of theinvention for target sequences that comprise DNA. In addition, Faruquidiscloses in U.S. Pat. No. 6,368,801 that T4 RNA ligase can efficientlyligate DNA ends of nucleic acids that are adjacent to each other whenhybridized to an RNA strand. Thus, T4 RNA ligase is a preferred ligaseof the invention in embodiments in which DNA ends are ligated on atarget sequence that comprises RNA. However, because of the highpotential for “background” ligation reactions, T4 RNA ligase is notpreferred when high specificity and/or high sensitivity is desired.Other ligases that ligate DNA ends of nucleic acids that are adjacent toeach other when hybridized to an RNA strand are also preferred fortarget nucleic acids comprising RNA. The invention is also not limitedto the use of a ligase for covalently joining target probe ends in thevarious embodiments of the invention. By way of example, other ligationmethods such as, but not limited to, topoisomerase-mediated ligation(e.g., U.S. Pat. No. 5,766,891, incorporated herein by reference) can beused, although topoisomerase-mediated ligation is not preferred in mostembodiments because of the high potential for background ligation. Insome other embodiments, chemical ligation methods can be used, such as,but not limited to, the use of a target probe with a 5′-end sequencethat comprises a 5′-iodo-nucleotide and a 3′-end comprising a nucleotidewith phosphorothioate, as disclosed by Xu, Y., and Kool, E. T. (NucleicAcids Res., 27: 875-881, 1999), which is incorporated herein byreference. The invention is not limited with respect to the ligationmethod used except that the ligation should occur efficiently in thepresence of a target sequence to which the target probes annealcontiguously and ligation should occur rarely or not at all in theabsence of a target sequence. As used herein, “ligation” refers to anysuitable method for joining adjacent 5′- and 3′-ends of target probesthat are adjacent or contiguous to each other when annealed to a targetsequence. In preferred ligation processes of the present invention, allof the target probes that anneal to a target sequence have a similarmelting temperature (T_(m)) with respect to the target sequence, and thelowest temperature at which a ligation process is performed is near theT_(m) of the target probe having the lowest T_(m) when it is annealed tothe target sequence.

[0379] K. Releasing Circular ssDNA Molecules that are Catenated to aTarget Sequence following Ligation Using the Target Sequence as aLigation Template

[0380] Bipartite probes that are ligated when annealed to a targetsequence create circular DNA molecules catenated to the target sequence(Nilsson, M. et al., Science, 265:2085-2088, 1994, incorporated hereinby reference). Nilsson et al. showed that, if the target sequence wasless than about 150-200 nucleotides from the 3′-end of the targetnucleic acid, the catenated circular ssDNA molecules obtained byligation of a linear probe on a target sequence were able to slip off ofthe target strand during denaturing washes, whereas circular moleculescatenated on the target sequence 850 nucleotides from the 3′-end of thetarget nucleic acid were not removed during denaturing washes.

[0381] The present invention comprises some embodiments in which a TSAcircle is replicated by rolling circle replication while catenated totarget nucleic acid or a target sequence tag using a DNA polymerase,such as but not limited to IsoTherm™ DNA polymerase (EPICENTRETechnologies, Madison, Wis.), Bst DNA polymerase large fragment, oranother DNA polymerase that can efficiently replicated catenatedtemplates. The present invention also comprises embodiments in whichcircular transcription substrates are transcribed by rolling circletranscription while they are catenated to a target nucleic acid ortarget sequence tag so long as the assay or method functions for itsintended purpose. It can be beneficial that the respective moleculesremain catenated to the target nucleic acid, and it is beneficial if thenumber of steps and the time to perform an assay is kept to the minimumto obtain the information for which the assay or method was intended.

[0382] However, in other embodiments, it can be desirable for a varietyof reasons that circular ssDNA ligation products obtained using a methodof the invention does not remain catenated to a target nucleic acid ortarget sequence tag comprising the target sequence following ligation.By way of example, but not of limitation, catenation of a TSA circleobtained by annealing and ligation of a target sequence amplificationprobe (TSA probe) on a target sequence or catenation of a circulartranscription substrate obtained by annealing and ligation of abipartite target probe on a target sequence and annealing of ananti-sense promoter oligo may limit the amount of rolling circlereplication product (e.g., see Baner, J. et al., Nucleic Acids Research,26: 5073-5078, 1998) or rolling circle transcription product,respectively, if the catenated circular molecules remain catenated tothe target nucleic acid or target sequence tag comprising the targetsequence following ligation.

[0383] However, the affect of catenation on the target nucleic acidshould be determined empirically in view of the results of Kuhn et al.(Nucleic Acids Res., 30: 574-580, 2002), incorporated herein byreference. Kuhn et al. showed that, although rolling circle replicationwas limited on catenated circular ssDNA molecules using phi29 DNApolymerase (which was used by Baner et al., Nucleic Acids Research, 26:5073-5078, 1998), the amount of rolling circle replication productobtained using catenated circular ssDNA molecules was not affected whenBst DNA polymerase large fragment, Sequenase® DNA polymerase (USB,Cleveland, Ohio), or Vent (exo-minus) DNA polymerase (New EnglandBiolabs, Beverly, Mass.) was used. Whether or not it is necessary torelease catenated circular ssDNA molecules from the target nucleic acidprior to rolling circle replication depends on the DNA polymerase used,indicating that the need to release catenated circular transcriptionsubstrates from the target nucleic acid may also depend on theparticular RNA polymerase used for rolling circle transcription.

[0384] Therefore, if a target nucleic acid comprising a target sequencedoes not have a free 3′-end that is less than about 150-200 nucleotidesfrom the target sequence, the present invention comprises empiricallydetermining if catenation of a ligation product obtained from ligationof a TSA probe or a bipartite target probe on the target sequenceresults in a reduction in the amount of product obtained during rollingcircle replication or rolling circle transcription, respectively,compared to the amount of product obtained on anoligodeoxyribo-nucleotide comprising only the target sequence. If theamount of product obtained is found to be decreased by catenation, thenan assay or method of the invention will either use additional steps torelease the catenated circular ssDNA molecule from the target nucleicacid for the particular assay or method, such as but not limited to oneof the methods to release catenated molecules described herein below, orwill use a different polymerase for which the amount of replicationproduct or transcription product is not affected by catenation.

[0385] In general, for this reason, a target sequence tag of the presentinvention will comprise a sequence that has a 3′-end that is less thanabout 150-200 nucleotides from the target sequence. Preferably, the3′-end of the target sequence tag is less than 100 nucleotides from thetarget sequence and most preferably, the 3′-end of the target sequencetag is less than 50 nucleotides from the target sequence.

[0386] With respect to a target sequence comprising a target nucleicacid in a sample, if the target sequence is more than about 150-200nucleotides from the 3′-end of the target nucleic acid, it is obvious toa person with knowledge in the art that there are a number of methodsfor breaking or cutting or shortening the target nucleic acid in orderto obtain a fragmented target nucleic acid comprising the targetsequence and any suitable method can be used to obtain a target nucleicacid for an assay or method of the present invention. Preferably, thetarget nucleic acid is fragmented to a size that has a 3′-end that isless than about 150-200 nucleotides from the target sequence prior touse of the target nucleic acid in an assay or method of the invention.

[0387] By way of example, but not of limitation, a DNA in a samplecomprising a dsDNA molecule or a ssDNA molecule to which an appropriatecomplementary DNA oligo is annealed can be digested with a restrictionendonuclease, provided that a suitable restriction site is presentwithin less than about 150-200 nucleotides from the 3′-end of the targetsequence and no restriction sites for the enzyme are present within thetarget sequence. Alternatively, if a suitable restriction site is notpresent on the target nucleic acid, one or more DNA oligonucleotideshaving a double-stranded segment that contains a FokI restriction enzymesite and a single-stranded segment that binds to the desired cleavagesite on a first-strand cDNA can be used. As is well known in the art,this type of oligonucleotide can be used with the restriction enzymeFokI to cut a single-stranded DNA at almost any desired sequence(Szybalski, W., Gene 40:169-173, 1985; Podhajska A. J. and Szybalski W.,Gene 40:175, 1985, incorporated herein by reference).

[0388] Still further, either RNA or DNA nucleic acids of known sequencecan be cleaved at specific sites using a 5′-nuclease or Cleavase™ enzymeand specific oligonucleotides, as described by Kwiatkowski, et al.,(Molecular Diagnosis 4:353-364, 1999) and in U.S. Pat. No. 6,001,567 andrelated patents assigned to Third Wave Technologies (Madison, Wis.,USA), which are incorporated herein by reference.

[0389] If the target nucleic acid is first-strand cDNA obtained byreverse transcription of RNA using a primer, the RNA can be cleaved withRNase H at a site to which a DNA oligo is annealed in order to definethe 3′-end of the reverse transcription product that is obtained.Alternatively, the length of the reverse transcription product can bekept within a desired size range by limiting the time of the reversetranscription reaction, which reverse transcription reaction can beoptimized for the particular primer, template sequence and reactionconditions used to obtain a target nucleic acid comprising a targetsequence, if present in the sample.

[0390] Still another method that can be used is to incorporate dUMPrandomly into the first-strand cDNA during reverse transcription orprimer extension to prepare a target nucleic acid comprising a targetsequence. In these embodiments, dUTP (deoxyribo-uridine triphosphate) isused in place of a portion of the dTTP (thymidine triphosphate) in thereaction. Also, dUTP can be incorporated in place of a portion of thedTTP in rolling circle replication of TSA circles that are used toincrease the number of target sequences available for annealing andligation of target probes for target-dependent transcription. Asdiscussed elsewhere herein, TSA circles are obtained by annealing andligation of target sequence amplification probes (TSA probes) on atarget sequence in a sample. When dUTP is used in a reversetranscription, primer extension or rolling circle replication reactionin addition to dTTP, dUMP will be incorporated randomly in place of dTMPat a frequency based on the ratio of dUTP to dTTP. Then, the respectivefirst-strand cDNA, primer extension product or rolling circlereplication product can be cleaved at sites of dUMP incorporation bytreatment (e.g., see U.S. Pat. No. 6,048,696, incorporated herein byreference) with uracil-N-glycosylase (UNG) and endonuclease IV (endoIV), which are available from EPICENTRE Technologies (Madison, Wis.,USA). UNG hydrolyzes the N-glycosidic bond between the deoxyribose sugarand uracil in single- and double-stranded DNA that contains uracil inplace of thymidine. UNG has no activity on dUTP or in cleaving uracilfrom UMP residues in RNA. Endo IV cleaves the phosphodiester linkage atthe abasic site. It may be useful to use a thermolabile UNG (e.g.,HK™-UNG from EPICENTRE Technologies, Madison, Wis., USA) for someapplications. (Also, incorporation of dUMP at one or more specific siteswithin a synthetic oligonucleotide introduces a specific cleavage sitewhich can be used at any time to cleave a resulting nucleic acid whichcontains the site by treatment with UNG and endo IV.)

[0391] Further, the 3′-end of a first-strand cDNA that is to become thetemplate sequence for a transcription reaction can be defined by firstamplifying the target nucleic acid sequence using any suitableamplification method, such as but not limited to PCR or RT-PCR, thatdelimits the end sequence.

[0392] If a 3′-end of a target sequence need not be at an exactlocation, and can be random or imprecise, which is the case in someembodiments of the invention, there are a number of other methods thatcan be used for making smaller fragments of a DNA molecule, whether fora target nucleic acid, a target sequence, or otherwise. By way ofexample, but not of limitation, a target nucleic acid can be fragmentedby physical means, such as by movement in and out of a syringe needle orother orifice or by sonication. If desired, the ends of physicallyfragmented double-stranded DNA can be made blunt prior to denaturationand use in an assay or method of the present invention using a T4 DNApolymerase or a kit, such as the End-It™ DNA End Repair Kit (EPICENTRETechnologies, Madison, Wis., USA).

[0393] Although it is preferred that a target nucleic acid comprising atarget sequence is short enough so that its 3′-end will easily bereleased from the catenated circular molecules that result from ligationof a bipartite target probe annealed to the target sequence, the presentinvention also includes embodiments of methods, assays, compositions andkits for detecting target sequences comprising larger target nucleicacids, wherein the catenated ligation product is not substantiallyreleased from the target nucleic acid. In those embodiments, theinvention comprises additional steps for release of the catenatedcircular ligation product after annealing and ligation on the targetsequence.

[0394] Baner, J. et al. (Nucleic Acids Research, 26: 5073-5078, 1998,incorporated herein by reference) showed that ligation of a linear DNAhaving two target-complementary end sequences that anneal adjacently ona target sequence resulted in a catenated molecule that was notefficiently replicated by rolling circle replication using phi29 DNApolymerase unless there was a free 3′-end of the target nucleic acidnear the ligation site. Baner et al. showed that, in order to obtainefficient rolling circle replication by phi29 DNA polymerase of circularssDNA molecules that had been ligated on a target sequence, thetopological link of the circular DNA with the target molecules needed tobe released. U.S. Pat. No. 6,558,928, incorporated herein by reference,provided methods for release of catenated circular DNA molecules inorder to improve the efficiency of rolling circle replication reactions.The present invention comprises the use of the methods described in U.S.Pat. No. 6,558,928, which methods are incorporated herein by reference,in order to release catenated circular ligation products for rollingcircle transcription as described herein.

[0395] In some embodiments, the circular ssDNA ligation product isreleased from catenation with the target sequence by digestion with anexonuclease after ligation of the bipartite probe on the targetsequence. Preferred exonucleases are those that digest single-strandedDNA and that do not have endonuclease activity. One enzyme that can beused is exonuclease I (exo I) (EPICENTRE Technologies, Madison, Wis.),which has 3′-to-5′ single-stranded exonuclease activity in the presenceof Mg²⁺ cations. Another enzyme that can be used is exonuclease VII (exoVII) (EPICENTRE Technologies, Madison, Wis.), which has both 3′-to-5′and 5′-to-3′ single-stranded exonuclease activity. Exo VII is active inthe absence of Mg²⁺ cations, which makes it a preferred embodiment formany applications. Rec J nuclease (EPICENTRE Technologies, Madison,Wis.), which has 5′-to-3′ single-stranded exonuclease activity in thepresence of Mg²⁺ cations, can also be used in some embodiments. Stillfurther, it is preferable to also use a double-stranded exonuclease,such as but not limited to exonuclease III (exo III) in addition to asingle-stranded exonuclease, such as but not limited to exo I, in orderto release catenated circular DNA molecules from a target nucleic acidcomprising a target sequence. In some embodiments, the target sequencethat has been digested with an exonuclease can prime rolling circlereplication after exonuclease removal of the non-base-paired 3′ end.Other methods in the art for releasing a catenated circular DNA moleculecan also be used.

[0396] L. DNA Polymerases and Processes of the Invention for Filling“Gaps” Between Target Probes

[0397] DNA polymerases are used in some embodiments of the presentinvention in order to fill by DNA polymerase extension one or more“gaps” between non-contiguous target-complementary sequences of targetprobes that are annealed to a target sequence. The invention is notlimited to a particular DNA polymerase to accomplish this purpose, andthe invention includes use of any DNA polymerase that is active infilling a gap under suitable reaction conditions. A suitable DNApolymerase fills the gap by DNA polymerase extension from the3′-hydroxyl end of one target-complementary target probe to the 5′-endof the next target-complementary target probe, withoutstrand-displacement of the target-complementary 5′-end portion of atarget probe. The “strand displacement” activity of a DNA polymerase isan operational definition and depends on reaction conditions, such as,but not limited to, reaction temperature, buffer, salt concentration,pH, Mg²⁺ concentration, use of cosolvents such as DMSO, or DNApolymerase enhancers such as betaine, as well as on the intrinsicproperties of a DNA polymerase. Thus, even though a particular DNApolymerase may have strand-displacement activity under certain reactionconditions, it may not have significant strand-displacement activityunder other reaction conditions. Thus, it is preferred that a DNApolymerase is evaluated for strand-displacement activity under thedesired reaction conditions of an assay or method of the invention.Strand displacement and DNA polymerase processivity can be assayed usingmethods described in Kong et al., (J. Biol. Chem., 268: 1965-1975,1993), which is incorporated herein by reference. Preferred DNApolymerases lack 5′-to′3′ and 3′-to-5′ exonuclease activity under thereaction conditions used. It is also important that the DNA polymeraseused to fill a gap lacks a 5′ structure-dependent nuclease, such asCleavase™ or Invader™ nucleases used by Third Wave Technologies(Madison, Wis.) because these enzymes could cleave off an unpairednucleotide, especially at the 5′-end of a sequence that is partiallyannealed to a target sequence 3′- of another target probe, and then theDNA polymerase could fill in the gap formed. Therefore, a DNA polymerasewith 5′ structure-dependent nuclease activity could result in inaccurateresults in the assay. Most preferred DNA polymerases are thermostable sothat activity is more consistent during the course of a method or assayof the invention and in order to be more easily stored without loss ofpolymerase activity. A preferred DNA polymerase of the invention forfilling gaps between target probes annealed to a target sequence is T4DNA polymerase. Another DNA polymerase that can be used is T7 DNApolymerase (Epicentre Technologies, Madison, Wis., USA).

[0398] M. Transcription Processes of the Invention

[0399] The target-complementary sequence at the 5′-end of a bipartitetarget probe or the 5′-end of a promoter target probe is a template fortranscription by the cognate RNA polymerase that recognizes thepromoter. However, since the target-complementary sequence at the 5′-endof a bipartite target probe of a promoter target probe is short,comprising only about 4-100 nucleotides, preferably about 8-30nucleotides, the reaction conditions of the method of the invention canbe adjusted so that RNA that is complementary to thetarget-complementary sequence at the 5′-end of a bipartite target probeor a promoter target probe, if it is synthesized by the RNA polymerase,forms an RNA:DNA hybrid and the RNA is not displaced from the DNAtemplate under the transcription reaction conditions used. Thus, in theabsence of a target sequence that permits ligation of thetarget-complementary sequence at the 5′-end of a bipartite target probeor a promoter target probe to the 3′-end of a secondtarget-complementary sequence, only a maximum of about one RNA copy ofthe target-complementary sequence at the 5′-end of the bipartite targetprobe or promoter target probe is synthesized by the RNA polymerase andno RNA copy is synthesized that is complementary to atarget-complementary sequence or a signal sequence of a signal targetprobe or the 3′-end of a bipartite target probe, since these lattersequences are not joined to the promoter. Therefore, a method of thepresent invention detects, directly or indirectly, synthesis of RNA thatis complementary to the target-complementary sequence at the 3′-end of abipartite target probe or that is 5′- of a promoter target probe, i.e.,that is, complementary to a simple target probe or a signal targetprobe. In preferred embodiments of the invention, a target sequence isdetected, directly or indirectly, by transcription of a signal sequenceportion of a circular or linear transcription substrate. A preferredsignal sequence portion of the invention comprises a sequence thatencodes a substrate for a replicase, and the target sequence is detectedby contacting the RNA replicase substrate encoded by the signal sequenceportion with a replicase that replicates the transcribed RNA that is areplicase substrate under replication conditions. However, the inventionalso comprises other embodiments that use other signal sequences or nosignal sequence, as discussed herein above.

[0400] Paul Lizardi discusses the optional use of a transcriptionpromoter in an open circle probe (“OCP”) for use in rolling circleamplification (“RCA”), as disclosed in U.S. Pat. Nos. 6,344,329;6,210,884; 6,183,960; 5,854,033; 6,329,150; 6,143,495; 6,316,229;6,287,824. However, in contrast to the methods of the present invention,Lizardi disclosed that a promoter portion can be included in an opencircle probe so that RNA transcripts can be generated from tandemsequence DNA (“TS-DNA”), which is a product of rolling circleamplification. In contrast, in the methods of the present invention, theRNA transcripts are primary amplification products and are synthesizedby in vitro transcription of transcription substrates obtained bytarget-dependent joining of target probes. Thus, the RNA transcripts ofthe present invention are complementary to the target probes used in anassay or method. Preferred promoters in the methods of Lizardi are T7 orSP6 RNA polymerase promoters, which are double-stranded promoters, andthe cognate polymerase for the promoter is used for transcriptionalamplification. Thus, in embodiments of Lizardi's invention that containa promoter sequence, Lizardi's open circle probes actually contain aprotopromoter sequence, to which a complementary sequence must beannealed or a second DNA strand needs to be synthesized in order toobtain a functional promoter. Lizardi further states that a promoter onan open circle probe, if present, is preferably immediately adjacent tothe left target probe (i.e., the promoter is 5′- of thetarget-complementary sequence on the 3′-end of the open circle probe)and is oriented to promote transcription toward the 3′-end of the opencircle probe so the orientation results in transcripts that arecomplementary to TS-DNA. Thus, the position and orientation of apromoter sequence in the methods disclosed by Lizardi are completelydifferent and would not be workable for the methods of the presentinvention. As discussed elsewhere herein, a single-stranded promoter ofthe present invention must be located 3′- of the target-complementarysequence at the 5′-end of a bipartite target probe or must be located3′- of the target-complementary sequence at the 5′-end of a monopartitepromoter target probe in order to function in the assays and methods ofthe invention. In short, promoters, if present at all, are included inopen circle probes for the methods of Lizardi in order to obtainsecondary amplification of DNA replication products, rather than for thepurpose of primary amplification as is the case in the methods andassays of the present invention. Also, the RNA polymerases of thepresent invention have not been used for amplification methods in theprior art. The RNA polymerases of the present invention lackhelicase-like activity and use single-stranded promoters and templatesfor transcription, whereas the T7-type RNA polymerases, such as T7 RNAPand SP6 RNAP, used by Lizardi and others have helicase-like activity forunwinding of dsDNA. Therefore, Lizardi and others were not able toenvision embodiments of the present invention that use single-strandedpromoters and templates.

[0401] The present invention also differs from and provides significantadvantages over the methods disclosed in Japanese Patent Nos. JP4304900and JP4262799 of Aono Toshiya et al. First, the method of Toshiya et al.disclosed use of an RNA polymerase with helicase-like activity, such asT7, T3 or SP6 RNA polymerase. In contrast, the novel mini-vRNApolymerases disclosed in the present invention lack helicase-likeactivity. Mini-vRNAP enzymes use single-stranded DNA templates and areunable to unwind or transcribe double-stranded DNA. Mini-vRNAP enzymesalso are unable to displace the RNA product from the RNA: DNA hybridobtained from transcription of linear templates. The RNA product from invitro transcription using mini-vRNAP is displaced from linear templatesonly in the presence of EcoSSB Protein. This lack of helicase-likeactivity and lack of activity in displacing the transcription productresults in low background transcription of the linear target probes inan assay or method of the current invention.

[0402] Still further, the method of Toshiya et al. requires annealing acomplementary nucleotide primer having an anti-promoter sequence inorder to form a functional double-stranded promoter for the RNApolymerase. In contrast, the RNA polymerases disclosed in the presentinvention use single-stranded promoters and single-stranded templates.Therefore, the present invention does not use a step for annealing of an“anti-promoter” sequence. As shown in Example 8, although we observed ahigh level of rolling circle transcription product using mini-vRNAP witha circular single-stranded transcription substrate having a P2 promoter,we did not detect mini-vRNAP transcription products using the unligatedprecursor comprising a linear single-stranded oligonucleotide having aP2 promoter sequence. Also, no transcription was detected with acircular single-stranded oligonucleotide that either lacked a P2promoter or had an anti-sense P2 promoter instead of the sense P2promoter.

[0403] It is known that large amounts of transcription product areobtained by in vitro transcription of short DNA sequences that arejoined to a functional double-stranded T7 RNAP promoter (Milligan, J Fet al., Nucleic Acids Res., 15: 8783-8798, 1987). Therefore, using themethod of Toshiya et al., the amount of transcription product obtainedduring in vitro transcription in the presence of unligated linear probeto which the anti-promoter primer was annealed would be significant andwould seriously limit the sensitivity of the method. Still further,based on the schematic diagrams provided in Japanese Patent Nos.JP4304900 and JP4262799, it did not appear that the double-strandedpromoter sequence in the method disclosed was positioned close to the5′-end of the linear probe in order to minimize transcription of thelinear template upon annealing of the anti-promoter primer.

[0404] These problems may explain why the methods disclosed in JapanesePatent Nos. JP4304900 and JP4262799 did not appear to have been pursued.The inventors believe that the methods of the present invention that usenovel mini-vRNAP enzymes and completely single-stranded transcriptionsubstrates having single-stranded promoters solve these problems.

[0405] N. Amplification and Detection Processes of the Invention:Amplifying a Target Sequence and Amplifying a Signal Sequence

[0406] The terms “amplifying a target” or “amplifying a target nucleicacid” or “amplifying a target nucleic acid sequence” or “amplifying atarget sequence” herein mean increasing the number of copies of thatportion of the sequence of a target nucleic acid for which acomplementary sequence is present in a target probe of the invention,including, but not limited to, a a target-complementary sequence that ispresent in a target probe that also comprises a sequence for atranscription promoter for an RNA polymerase. An “amplified target” oran “amplified target sequence” comprises only that portion of thesequence of a target nucleic acid for which a complementary sequence ispresent in a target probe of the invention. The use of the terms“amplifying a target” or “amplifying a target nucleic acid” or“amplifying a target nucleic acid sequence” or “amplifying a targetsequence” herein is not intended to imply that all of the sequence of atarget nucleic acid is amplified. The use of these terms is also notintended to imply that the amplification of that portion of the sequenceof a target nucleic acid for which a complementary sequence is presentin a target probe of the invention is actually directly observed ordetected in a method or assay of the invention. The invention comprisesembodiments in which the amplified target sequence is directly detected,such as, but not limited to, embodiments in which the target sequence isdetected by measuring a fluorescent signal following annealing of atranscript-complementary detection probe such as, but not limited to amolecular beacon. The invention also comprises embodiments in which theamplified target sequence is detected only indirectly by generation ofanother signal, such as, but not limited to, embodiments in which asignal is generated as a result of transcription of another DNA sequencethat is covalently attached to a target-complementary sequence and thatis transcribed along with a target-complementary sequence. By way ofexample, but not of limitation, in one embodiment, which is a preferredembodiment, the amplification of a target sequence is detected bydetecting a substrate for Q-beta replicase. The substrate is replicatedby Q-beta replicase using replication conditions well known in the artfollowing synthesis of said RNA substrate by transcription of a signalsequence portion of a target probe that encodes said Q-beta substrate.The term “amplification signal” as used herein is intended to describethe output or result of any method, whether direct or indirect, fordetecting if amplification of a target sequence has occurred. By way ofexample, but not of limitation, an amplification signal can comprise afluorescent signal that results from annealing of a molecular beacon toan RNA transcript that is complementary to a target probe, or anamplification signal can comprise a Q-beta substrate that is replicatedby Q-beta replicase following transcription of a DNA portion of a targetprobe that encodes said Q-beta substrate. As discussed previously withrespect to signal sequences, the invention comprises any signal sequenceand any detection method that detects target-dependent transcription ofa target sequence or a signal sequence.

[0407] O. Reverse Transcriptases and Reverse Transcription Processes ofthe Invention

[0408] In some embodiments in which a target nucleic acid in a samplecomprises RNA, reverse transcription is used to obtain a target sequencecomprising DNA. Also, some embodiments of methods and assays of thepresent invention use reverse transcription processes in conjunctionwith other processes in order to obtain additional amplification of atarget sequence and/or a signal sequence. These embodiments use areverse transcriptase. A “reverse transcriptase” or “RNA-dependent DNApolymerase” is an enzyme that synthesizes a complementary DNA copy(“cDNA”) from an RNA template. All known reverse transcriptases alsohave the ability to make a complementary DNA copy from a DNA template;thus, they are both RNA- and DNA-dependent DNA polymerases. A primer isrequired to initiate synthesis with both RNA and DNA templates. Examplesof reverse transcriptases that can be used in methods of the presentinvention include, but are not limited to, AMV reverse transcriptase,MMLV reverse transcriptase, Tth DNA polymerase, rBst DNA polymeraselarge fragment, also called IsoTherm™ DNA Polymerase (EpicentreTechnologies, Madison, Wis., USA), and BcaBEST™ DNA polymerase (TakaraShuzo Co, Kyoto, Japan). In some cases, a mutant form of a reversetranscriptase, such as, but not limited to, an AMV or MMLV reversetranscriptase that lacks RNase H activity can be used. In otherembodiments, a wild-type enzyme is preferred. In some embodiments of theinvention, a separate RNase H enzyme, such as but not limited to, E.coli RNase H or Hybridase™ Thermostable RNase H (Epicentre Technologies,Madison, Wis. 53713, USA). can also be used in reverse transcriptionreactions. MMLV reverse transcriptase (wild-type, RNase H-positive) ispreferred for some embodiments of the invention in which it can be usedwithout a separate RNase H enzyme. In some other embodiments, IsoTherm™DNA polymerase or AMV reverse transcriptase can be used. The processesof the invention include conducting experiments to determine the effectson amplification of RNase H activity of a reverse transcriptase and/orseparate RNase H enzyme(s) used, including, but not limited to, AMVreverse transcriptase, IsoTherm DNA polymerase, and both RNase H-plusand RNase H-minus MMLV reverse transcriptase, and E. coli RNase H orthermostable RNase H enzymes that are stable for more than 10 minutes at70° C. (U.S. Pat. Nos. 5,268,289; 5,459,055; and 5,500,370), such as,but not limited to, Hybridase™ thermostable RNase H, Tth RNase H, andTfl RNase H (Epicentre Technologies, Madison, Wis., USA), or bydifferent combinations of a reverse transcriptase and a separate RNaseH. Kacian et al. (U.S. Pat. No. 5,399,491), incorporated herein byreference, discloses information related to the effects of addingdifferent amounts of a separate RNase H enzyme to transcription-mediatedamplification assays that used T7 RNAP and dsDNA templates and eitherMMLV or AMV reverse transcriptase, which information is useful insuggesting how to vary and evaluate reaction conditions related to useof reverse transcriptases and RNase H enzymes in methods and assays ofthe present invention.

[0409] P. Strand-Displacing DNA Polymerases for Rolling CircleReplication Processes of the Invention

[0410] Some DNA polymerases are able to displace the strandcomplementary to the template strand as a new DNA strand is synthesizedby the polymerase. This process is called “strand displacement” and theDNA polymerases that have this activity are referred to herein as“strand-displacing DNA polymerases.” If the DNA template is asingle-stranded circle, primed DNA synthesis procedes around and aroundthe circle, with continual displacement of the strand ahead of thereplicating strand, a process called “rolling circle replication.”Rolling circle replication results in synthesis of tandem copies of thecircular template. The suitability of a DNA polymerase for use in anembodiment of the invention that comprises rolling circle replicationcan be readily determined. By way of example, but not of limitation, theability of a polymerase to carry out rolling circle replication can bedetermined by using the polymerase in a rolling circle replication assayas described by Fire and Xu (Proc. Natl. Acad. Sci. USA, 92: 4641-4645,1995), incorporated herein by reference. It is preferred that a DNApolymerase be a strand displacing DNA polymerase and lack a 5′-to-3′exonuclease activity for strand displacement polymerization reactionsusing both linear or circular templates since a 5′-to-3′ exonucleaseactivity, if present, might result in the destruction of the synthesizedstrand. It is also preferred that DNA polymerases for use in thedisclosed strand displacement synthesis methods are highly processive.The ability of a DNA polymerase to strand-displace can vary withreaction conditions, in addition to the particular enzyme used. Stranddisplacement and DNA polymerase processivity can also be assayed usingmethods described in Kong et al (J. Biol. Chem., 268: 1965-1975, 1993and references cited therein, all of which are incorporated herein byreference).

[0411] Preferred strand displacing DNA polymerases of the invention areRepliPHI™ phi29 DNA polymerase (EPICENTRE Technologies, Madison, Wis.,USA), phi29 DNA polymerase, rBst DNA polymerase large fragment (alsocalled IsoTherm™ DNA polymerase (EPICENTRE Technologies, Madison, Wis.,USA), BcaBEST™ DNA polymerase (Takara Shuzo Co., Kyoto, Japan), andSequiTherm™ DNA polymerase (EPICENTRE Technologies, Madison, Wis., USA).Other strand-displacing DNA polymerases which can be used include, butare not limited to phage M2 DNA polymerase (Matsumoto et al., Gene, 84:247, 1989), phage phi PRD1 DNA polymerase (Jung et al., Proc. Natl.Acad. Sci. USA, 84: 8287, 1987), VENT® DNA polymerase (Kong et al., J.Biol. Chem. 268: 1965-1975, 1993), Klenow fragment of DNA polymerase I(Jacobsen et al., Eur. J. Biochem. 45: 623-627, 1974), T5 DNA polymerase(Chatterjee et al., Gene 97:13-19, 1991), PRD1 DNA polymerase (Zhu andIto, Biochim. Biophys. Acta, 1219: 267-276, 1994), or T7 DNA polymerasein the presence of a T7 helicase/primase complex (Tabor and Richardson,Abstact No. 11, presented at the meeting “New Horizons in Genomics,”Mar. 30-Apr. 1, 2003 in Santa Fe, New Mexico, sponsored by the DOE JointGenome Institute. Strand displacing DNA polymerases are also useful insome embodiments of the invention for strand displacement replication oflinear first-strand cDNA, and in other embodiments, for rolling circlereplication of circular first-strand cDNA.

[0412] In general, it is desirable that the amount of strand-displacingDNA polymerase in the reaction be as high as possible without inhibitingthe reaction. By way of example, but without limitation, RepliPHI™ phi29DNA Polymerase can be used at about one microgram of protein in a20-microliter reaction and IsoTherm™ DNA Polymerase can be used at about50 units to about 300 units in a 50-microliter reaction. Sincedefinitions for units vary for different DNA polymerases and even forsimilar DNA polymerases from different vendors or sources, and alsobecause the activity for each enzyme varies at different temperaturesand under different reaction conditions, it is desirable to optimize theamount of strand-displacing DNA polymerase and reaction conditions foreach target sequence and particular assay or method of the invention.

[0413] Although not required for all DNA polymerases, stranddisplacement can be facilitated for some DNA polymerases through the useof a strand displacement factor, such as a helicase. It is consideredthat any DNA polymerase that can perform rolling circle replication inthe presence of a strand displacement factor is suitable for use inembodiments of the invention that comprise rolling circle replication,even if the DNA polymerase does not perform rolling circle replicationin the absence of such a factor. Strand displacement factors useful inrolling circle replication include, but are not limited to, BMRF1polymerase accessory subunit (Tsurumi et al., J. Virology, 67:7648-7653, 1993), adenovirus DNA-binding protein (Zijderveld and van derVliet, J. Virology, 68: 1158-1164, 1994), herpes simplex viral proteinICP8 (Boehmer and Lehman, J. Virology, 67: 711-715, 1993); Skaliter andLehman, Proc. Natl. Acad. Sci. USA, 91: 10,665-10,669, 1994),single-stranded DNA binding proteins (SSB; Rigler and Romano, J. Biol.Chem., 270: 8910-8919, 1995), and calf thymus helicase (Siegel et al.,J. Biol Chem., 267: 13,629-13,635, 1992).

[0414] Q. Methods and Assays of the Invention for Detecting a TargetSequence

[0415] The present invention comprises methods, compositions and kitsfor detecting one or multiple specific target sequences in a sample bytarget-dependent transcription. FIG. 20 shows one basic embodiment of amethod of the present invention. This embodiment uses a bipartite targetprobe. A bipartite target probe is a linear single-stranded DNA moleculethat has sequences on both ends of the probe that are complementary todifferent portions of a target sequence. In the embodiment shown in FIG.20, the target-complementary sequences of the bipartite target probe arecontiguous or adjacent or abut to each other when annealed to the targetsequence. The sequence at the 5′-end of the bipartite target probepreferably has a 5′-phosphate group or is phosphorylated by apolynucleotide kinase during the course of a method of the invention.The 5′-portion of the bipartite target probe also has a sequence for asingle- stranded transcription promoter that is a functional promoterfor a DNA-dependent RNA polymerase that can bind to this single-strandedpromoter and initiate transcription of RNA therefrom in a 5′-to-3′direction using single-stranded DNA that is 5′- of and covalently linkedto the promoter as a template. The promoter is oriented within thesingle-stranded DNA of the bipartite target probe 3′- of thetarget-complementary sequence at the 5′-end of the 5′-portion. Thesequence at the 3′-end of the bipartite target probe preferably has a3′-hydroxyl group.

[0416] Referring to FIG. 20, in the presence of a a target sequencecomprising a single-stranded DNA target or one strand of adouble-stranded DNA target, a bipartite target probe anneals to thetarget sequence under hybridization conditions, wherein the5′-phosphorylated end of the bipartite target probe is adjacent to its3′-hydroxyl end. Then, the ends of the bipartite target probe areligated under ligation conditions by contacting the target-complementaryends annealed to a target sequence with a ligase that has little or noactivity in ligating free ends that are not annealed to a complementarysequence but is active in joining a 5′-phosphorylated end to a3′-hydroxylated end when the ends are adjacent when annealed to acomplementary DNA sequence. Ligation of the ends of the bipartite targetprobe generates a “circular transcription substrate,” meaning a circularsingle-stranded DNA molecule that is a template for transcription by anRNA polymerase that recognizes a promoter sequence in said circulartranscription substrate.

[0417] Thus, again referring to FIG. 20, one embodiment of the presentinvention comprises a method for detecting a target sequence, saidmethod comprising:

[0418] a. providing a bipartite target probe, wherein said bipartitetarget probe comprises a linear single-stranded DNA (ssDNA) comprisingtwo end portions that are complementary to a contiguous target sequence,and wherein said bipartite target probe forms a circular transcriptionsubstrate upon joining of said ends;

[0419] b. annealing said bipartite target probe to said target nucleicacid comprising said target sequence under hybridization conditions;

[0420] c. ligating said bipartite target probe annealed to said targetnucleic acid under ligation conditions with a ligase, wherein saidligase has little or no activity in ligating blunt ends and issubstantially more active in ligating said ends of said bipartite targetprobe if said ends are adjacent when annealed to two contiguous regionsof a target sequence than if said ends are not annealed to said targetsequence, so as to obtain a circular ssDNA molecule that comprises acircular transcription substrate;

[0421] d. obtaining said circular transcription substrate, wherein saidcircular transcription substrate comprises a sequence that iscomplementary to said target sequence;

[0422] e. contacting said circular transcription substrate with an RNApolymerase under transcription conditions so as to synthesize RNA thatis complementary to said circular transcription substrate; and

[0423] f. detecting the synthesis of RNA resulting from transcription ofsaid circular transcription substrate, wherein said synthesis of saidRNA indicates the presence of said target sequence comprising saidtarget nucleic acid.

[0424] Also, since transcription of said circular transcriptionsubstrate increases the number of copies of the target sequence, theinvention also comprises a method for amplifying a target sequence, saidmethod comprising:

[0425] a. providing a bipartite target probe, wherein said bipartitetarget probe comprises a linear single-stranded DNA (ssDNA) comprisingtwo end portions that are complementary to a contiguous target sequence,wherein said bipartite target probe forms a circular transcriptionsubstrate upon joining of said ends;

[0426] b. annealing said bipartite target probe to said target nucleicacid comprising said target sequence under hybridization conditions;

[0427] c. ligating said bipartite target probe annealed to said targetnucleic acid under ligation conditions with a ligase, wherein saidligase has little or no activity in ligating blunt ends and issubstantially more active in ligating said ends of said bipartite targetprobe if said ends are adjacent when annealed to two contiguous regionsof a target sequence than if said ends are not annealed to said targetsequence, so as to obtain a circular ssDNA molecule that comprises acircular transcription substrate;

[0428] d. obtaining said circular transcription substrate, wherein saidsubstrate comprises a sequence that is complementary to said targetsequence;

[0429] e. contacting said circular transcription substrate with an RNApolymerase under transcription conditions so as to synthesize RNA thatis complementary to said circular transcription substrate; and

[0430] f. obtaining RNA transcripts comprising multiple copies of saidtarget sequence.

[0431]FIG. 21 shows an embodiment of a method or assay of the inventionthat is similar to the embodiment shown in FIG. 20 except that thebipartite target probe used in the method shown in FIG. 21 does not havea transcription termination sequence and transcription of the circulartranscription substrate resulting therefrom generates a transcriptionproduct comprising an RNA multimer by rolling circle transcription.

[0432] Thus, referring to FIG. 21, one embodiment of the presentinvention comprises a method for detecting a target sequence, saidmethod comprising:

[0433] a. providing a bipartite target probe, wherein said bipartitetarget probe comprises a linear single-stranded DNA comprising two endportions that are complementary to a contiguous target sequence, andwherein said bipartite target probe forms a circular transcriptionsubstrate upon joining of said ends;

[0434] b. annealing said bipartite target probe to said target nucleicacid comprising said target sequence under hybridization conditions;

[0435] c. ligating said bipartite target probe annealed to said targetnucleic acid under ligation conditions with a ligase, wherein saidligase has little or no activity in ligating blunt ends and issubstantially more active in ligating said ends of said bipartite targetprobe if said ends are adjacent when annealed to two contiguous regionsof a target sequence than if said ends are not annealed to said targetsequence, so as to obtain a circular ssDNA molecule that comprises acircular transcription substrate;

[0436] d. obtaining said circular transcription substrate, wherein saidcircular transcription substrate comprises a sequence that iscomplementary to said target sequence;

[0437] e. contacting said circular transcription substrate with an RNApolymerase under rolling circle transcription conditions so as tosynthesize RNA multimers, wherein an RNA multimer comprises multipletandem copies of an oligomer that is complementary to one copy of saidcircular transcription substrate; and

[0438] f. detecting the synthesis of said RNA resulting from rollingcircle transcription of said circular transcription substrate, whereinsaid synthesis of said RNA indicates the presence of said targetsequence comprising said target nucleic acid.

[0439] Other embodiments of the present invention, as shown in FIG. 22,comprise compositions, methods and kits for detecting one or multiplespecific target sequences in a sample by coupled target-dependentrolling circle replication (RCR) and rolling circle transcription (RCT).These embodiments use bipartite target probes to generate circulartranscription substrates as shown in either FIG. 20 or FIG. 21, whichresult in circular transcription substrate either with a transcriptionterminator or lacking a transcription terminator, respectively. If thecircular transcription substrate lacks a transcription terminatorsequence, transcription comprises rolling circle transcription asdescribed elsewhere herein. In addition to using a bipartite targetprobe, these embodiments also use a “bipartite target sequenceamplification probe,” which is also referred to as a “bipartite TSAprobe” or simply as a “TSA probe” herein. The purpose of a TSA probe inan assay or method is to obtain a target-dependent amplification of thenumber of copies of the target sequence and thereby, to provideadditional sites for annealing and ligation of the bipartite targetprobe.

[0440] A TSA probe is a linear single-stranded DNA molecule thatcomprises two target-complementary sequences that are connected by anintervening sequence that is not complementary to the target sequence.The target-complementary portions on the ends are complementary todifferent portions of a target sequence in a target nucleic acid or atarget sequence tag of an analyte-binding substance. Each of the 5′ and3′ target-complementary sequences in a TSA probe for a particular assayor method is identical to the corresponding target-complementarysequence at the 5′-end or the 3′-end of a bipartite target probe used inthe assay or method. That is, the 5′-end of a TSA probe anneals to thesame nucleotides in the target sequence as the 5′-end of thecorresponding bipartite target probe that is used to obtain a circulartranscription substrate and similarly, the 3′-end of the TSA probeanneals to the same nucleotides of the target sequence as the 3′-end ofthe bipartite target probe. Thus, as shown in FIG. 22, thetarget-complementary sequences of the TSA probe are adjacent to eachother when annealed to the target sequence in exactly the same manner asdescribed previously for bipartite target probes. Similarly, thesequence at the 5′-end of the TSA probe preferably has a 5′-phosphategroup or is phosphorylated by a polynucleotide kinase during the courseof a method of the invention and the sequence at the 3′-end of a TSAprobe preferably has a 3′-hydroxyl group. After annealing to a targetsequence, if present in a sample, the adjacent target-complementarysequences of a TSA probe are ligated in a method of the invention with aligase that has little or no activity in ligating blunt ends and that issubstantially more active in ligating said ends that are adjacent whenannealed to two contiguous regions of a target sequence than if saidends are not so annealed. Ligation of a TSA probe results in formationof a “TSA circle,” which, upon annealing to a primer, is a substrate forrolling circle replication.

[0441] The target-complementary sequences of a TSA probe are connectedby an intervening sequence. The sequence and nucleotide composition ofthe intervening sequence can vary, but it should comprise a sequence ofsufficient length and sequence specificity to provide a primer-bindingsite for specific priming by a primer for rolling circle replication.The intervening sequence should also be of sufficient length to permitthe target-complementary sequences of the TSA probe to anneal to thetarget sequence with specificity. In addition, the length of theintervening sequence should be optimized to obtain the optimaltarget-dependent ligation efficiency with the ligase and the maximumrolling circle replication rate and maximum end-point level of RCRproduct with the strand-displacing DNA polymerase under the assayconditions used. Although a bipartite target probe could also be used asa TSA probe, it is preferable that the TSA probe is not a bipartitetarget probe. Preferably, a TSA probe does not have a transcriptionpromoter sequence, a transcription termination sequence, or a signalsequence, and preferably the primer-binding site in a TSA probe for astrand-displacing DNA polymerase primer used for rolling circlereplication is not present in the corresponding bipartite target probe.The lack of a promoter sequence in the TSA probe or the resulting TSAcircle permits maximum rolling replication because there is no promoterto bind an RNA polymerase or initiate transcription. Similarly, the lackof a primer-binding site for priming by a strand-displacing DNApolymerase on the bipartite target probe or the resulting circulartranscription substrate permits maximum transcription because there isnot site for priming a competitive rolling circle transcriptionreaction.

[0442] Referring to FIG. 22, in the presence of a target sequence, a TSAprobe anneals to the target sequence under hybridization conditions,wherein the 5′-phosphorylated end of the TSA probe is adjacent to its3′-hydroxyl end. Then, the ends of the TSA probe are ligated underligation conditions by contacting the target-complementary ends annealedto a target sequence with a ligase that has little or no activity inligating free ends that are not annealed to a complementary sequence butis active in joining a 5′-phosphorylated end to a 3′-hydroxylated endwhen the ends are adjacent when annealed to a complementary DNAsequence. Ligation of the ends of the TSA probe generates a TSA circle.Upon annealing of a primer to the TSA circle and contacting theresulting complex with a strand-displacing DNA polymerase understrand-displacing polymerization conditions, rolling circle replicationoccurs, thereby generating multiple tandem copies of the target sequenceto which the target-complementary sequences of a bipartite target probecan anneal under hybridization conditions. The adjacent5′-phosphorylated end and the 3′-hydroxyl end of the bipartite targetprobes annealed to the tandem target sequences of the rolling circlereplication products are ligated by the ligase under ligationconditions, thereby generating a circular transcription substrate.Transcription products are obtained by contacting the circulartranscription substrates with an RNA polymerase that can bind thesingle-stranded promoter and initiate transcription therefrom using asingle-stranded template, and the transcription products are obtained ordetected by a suitable means.

[0443] Thus, again referring to FIG. 22, one embodiment of the presentinvention comprises a method for obtaining a transcription productcomplementary to a target nucleic acid sequence (target sequence), saidmethod comprising:

[0444] a. providing a TSA probe, wherein said TSA probe comprises alinear single-stranded DNA (ssDNA) comprising two end portions that arecomplementary to the contiguous target sequence that are connected by anintervening sequence, and wherein said TSA probe can form a TSA circleupon joining of said ends;

[0445] b. providing a primer that is complementary to the interveningsequence of said TSA probe;

[0446] c. providing a bipartite target probe, wherein said bipartitetarget probe comprises a linear ssDNA comprising two end portions thatare complementary to a contiguous target sequence, and wherein saidbipartite target probe forms a circular transcription substrate uponjoining of said ends;

[0447] d. annealing said TSA probe to said target sequence underhybridization conditions;

[0448] e. ligating said TSA probe annealed to said target sequence underligation conditions with a ligase, wherein said ligase has little or noactivity in ligating blunt ends and is substantially more active inligating said ends of said bipartite target probe if said ends areadjacent when annealed to two contiguous regions of a target sequencethan if said ends are not annealed to said target sequence, so as toobtain a TSA circle;

[0449] f. annealing the primer that is complementary to the interveningsequence of the TSA probe to the TSA circle under hybridizationconditions;

[0450] g. contacting said TSA circle to which said primer is annealedwith a strand-displacing DNA polymerase under strand-displacingpolymerization conditions so as to obtain a rolling circle replicationproduct comprising multiple copies of the target sequence;

[0451] h. annealing said bipartite target probe to said multiple copiesof the target sequence of said rolling circle replication product underhybridization conditions;

[0452] i. ligating said bipartite target probe annealed to said multiplecopies of the target sequence of said rolling circle replication productwith a ligase under ligation conditions, wherein said ligase has littleor no activity in ligating blunt ends and is substantially more activein ligating ends that are adjacent when annealed to two contiguousregions of a target sequence than if said ends are not annealed, so asto obtain a circular ssDNA molecule that comprises a circulartranscription substrate;

[0453] j. obtaining said circular transcription substrate, wherein saidcircular transcription substrate comprises a sequence that iscomplementary to said target sequence;

[0454] k. contacting said circular transcription substrate with an RNApolymerase under transcription conditions so as to obtain atranscription product that is complementary to said circulartranscription substrate; and

[0455] l. obtaining said transcription product that is complementary tosaid circular transcription substrate, wherein said transcriptionproduct indicates the presence of said target sequence.

[0456] Preferably, only one ligase is used in this embodiment forligating both the TSA probe and the bipartite target probe. Preferably,the ligase has little or no activity in ligating blunt ends and issubstantially more active in ligating ends that are adjacent whenannealed to two contiguous regions of a target sequence compared to endsthat are not annealed to the target sequence. One suitable ligase thatcan be used is Ampligase® Thermostable DNA Ligase (EPICENTRETechnologies, Madison, Wis.). A preferred strand-displacing DNApolymerase that can be used is IsoTherm™ DNA Polymerase (EPICENTRETechnologies, Madison, Wis.). Another suitable strand-displacing DNApolymerase that can be used is RepliPHI™ phi29 DNA polymerase (EPICENTRETechnologies, Madison, Wis.). Some preferred RNA polymerases are T7RNAP, T3 RNAP, SP6 RNAP or another T7-like RNA polymerase or a mutantform of one of these T7-like RNA polymerases. Preferably, AmpliScribeT7-Flash™ Transcription Kit is used for in vitro transcription of thetranscription substrate (EPICENTRE Technologies, Madison, Wis.).

[0457] In some embodiments, the target sequence comprises a targetnucleic acid in a sample, whereas in other embodiments the targetsequence comprises a target sequence tag that is joined to ananalyte-binding substance that binds an analyte in the sample. In someembodiments, the TSA circle that is replicated remains catenated to atarget nucleic acid. Preferably, the target sequence is less than about150 to about 200 nucleotides from the 3′-end of the target nucleic acidor target sequence tag. In other embodiments of methods in which thetarget sequence is greater than about 150 to about 200 nucleotides fromthe 3′-end of the target nucleic acid or target sequence tag, then oneor more additional steps is used in order to release the catenated TSAcircles from the target sequence prior to rolling circle replication, asdescribed elsewhere herein. Similarly, one or more additional steps canbe used in order to release the catenated circular ssDNA ligationproducts (which are circular transcription substrates) that result fromligation of bipartite target probes that are annealed to targetsequences in the rolling circle replication product more than about 150nucleotides to about 200 nucleotides from the 3′-end of to the rollingcircle replication product. In one preferred embodiment, rolling circlereplication is carried out using a ratio of dUTP to dTTP that results inincorporation of a dUMP residue about every 100-400 nucleotides and acomposition comprising uracil-N-glycosylase and endonuclease IV is usedto release catenated DNA molecules that are ligated on the linearrolling circle replication product following annealing of bipartitetarget probes to the replicated target sequences.

[0458]FIG. 23 shows one aspect of another embodiment of a method of thepresent invention. This embodiment also uses a bipartite target probethat is similar to a bipartite target probe used in the method shown inFIG. 20, except that the target-complementary sequences of the bipartitetarget probe used in the embodiment shown in FIG. 23 are not contiguousor adjacent to each other when annealed to the target sequence. Rather,the target-complementary sequences of a bipartite target probe of thisembodiment are separated from each other when they are annealed to thetarget sequence. The gap between the two target-complementary sequencescan comprise from about four nucleotides to about 1000 nucleotides ormore. Although the invention is not limited to a particular distancebetween the target-complementary sequences when annealed to a targetsequence, preferably the gap in this embodiment of the inventioncomprises from about six nucleotides to about 100 nucleotides, and mostpreferably, the gap comprises from about six nucleotides to about 25nucleotides. As in the previous embodiments, the 5′-end of the bipartitetarget probe in the embodiment in FIG. 23 preferably has a 5′-phosphategroup or is phosphorylated by a polynucleotide kinase during the courseof a method of the invention, and the 3′-end preferably has a3′-hydroxyl group. Also as in previously discussed embodiments, the5′-portion of the bipartite target probe in the embodiment of FIG. 23has sequence for a single-stranded transcription promoter that is afunctional promoter for a DNA-dependent RNA polymerase that can bind tothis single-stranded promoter and initiate transcription of RNAtherefrom in a 5′-to-3′ direction using single-stranded DNA which is 5′-of and covalently linked to the promoter. The promoter is orientedwithin the single-stranded DNA of a bipartite target probe 3′- of thetarget-complementary sequence at the 5′-end of the 5′-portion.

[0459] In the aspect of the embodiment of the method shown in FIG. 23,the gap between the target-complementary sequences of a bipartite targetprobe annealed to a target sequence is filled by also annealing one ormore simple target probes comprising target-complementary sequences thatanneal to the target sequence between portions of the target to whichthe target-complementary sequences of the bipartite target probe anneal.The simple target probes used anneal to the target sequence so as tofill the gap completely so as to abut with or to be contiguous with eachother and with the target-complementary sequences of the bipartitetarget probe. All 5′-ends of simple target probes and of the bipartitetarget probe have a 5′-phosphate group and all 3′-ends have hydroxylgroups. Thus, ligation of the bipartite target probe and simple targetprobes that are annealed to a target sequence with a ligase, whichligase has little or no activity in ligating free ends that are notannealed to a complementary sequence but is active in joining a5′-phosphorylated end to an adjacent 3′-hydroxylated end when the endsare annealed to a complementary DNA sequence, generates a circulartranscription substrate. Transcription of the circular transcriptionsubstrate results in synthesis of RNA that is complementary to thecircular transcription substrate and that can be used to detect thepresence of the target sequence.

[0460] Thus, again referring to FIG. 23, one embodiment of the presentinvention comprises a method for detecting a target sequence, saidmethod comprising:

[0461] a. providing a bipartite target probe, wherein said bipartitetarget probe comprises a linear single-stranded DNA (ssDNA) comprisingtwo end portions that are complementary to different non-contiguous 5′-and 3′-end portions of a target sequence;

[0462] b. providing one or more simple target probes, wherein saidsimple target probes are complementary to the target sequence so as toanneal to said target sequence in the gap between thetarget-complementary sequences of said bipartite target probe so as tocompletely fill said gap and so that each of the ends of said simpletarget probes are contiguous with an end of a simple target probe orwith an end of said bipartite target probe;

[0463] c. annealing said bipartite target probe and said simple targetprobes to said target sequence comprising said target nucleic acid underhybridization conditions;

[0464] d. ligating said bipartite target probe and said simple targetprobes annealed to said target sequence under ligation conditions with aligase, wherein said ligase has little or no activity in ligating bluntends and is substantially more active in ligating said ends of saidsimple target probes and said ends of said bipartite target probe ifsaid ends are adjacent when annealed to two contiguous regions of atarget sequence than if said ends are not annealed to said targetsequence, so as to obtain a circular ssDNA molecule that comprises acircular transcription substrate;

[0465] e. obtaining said circular transcription substrate, wherein saidsubstrate comprises a sequence that is complementary to said targetsequence;

[0466] f. contacting said circular transcription substrate with an RNApolymerase under transcription conditions so as to synthesize RNA thatis complementary to said circular transcription substrate; and

[0467] g. detecting the synthesis of RNA resulting from transcription ofsaid circular transcription substrate, wherein said synthesis of saidRNA indicates the presence of said target sequence comprising saidtarget nucleic acid.

[0468]FIG. 24 shows another aspect of an embodiment of a method of thepresent invention that uses a bipartite target probe that comprisestarget-complementary sequences that are separated from each other whenthey are annealed to a target sequence. However, in this embodiment, thegap between the target-complementary sequences of a bipartite targetprobe annealed to a target sequence is filled by primer extension usinga DNA polymerase and subsequently joined by ligation with a ligase ifand only if both the 3′-end of the target probe that is annealed to thetarget sequence 3′- of the gap and the target probe that is annealed tothe target sequence 5′- of the gap are complementary to and correctlybasepaired with the target sequence. If the 3′-end of the target probethat is 3′- of the gap is not annealed to the target sequence, then theDNA polymerase will be unable to fill the gap by primer extension. Also,if the 5′-end of the target probe that is 5′- of the gap is not annealedto the target sequence, then the 3′-end of the primer extension productwill not be adjacent to a 5′-end on the target sequence and it will notbe possible to join the 3′-end of the primer-extended target probe withthe 5′-phosphorylated end of the target probe annealed 5′- of the gap.

[0469] The gap between the two target-complementary sequences cancomprise from one nucleotide to about 1000 nucleotides or more. Althoughthe invention is not limited to a particular distance between thetarget-complementary sequences when annealed to a target sequence,preferably the gap comprises from one nucleotide to about 100nucleotides, and most preferably, the gap in most embodiments comprisesfrom one nucleotide to about 25 nucleotides. The 5′-end of a bipartitetarget probe in the embodiment in FIG. 24 preferably has a 5′-phosphategroup or is phosphorylated by a polynucleotide kinase during the courseof a method of the invention, and the 3′-end preferably has a3′-hydroxyl group. Also, the 5′-portion of the bipartite target probe inthe embodiment of FIG. 24 has sequence for a single-strandedtranscription promoter that is a functional promoter for a DNA-dependentRNA polymerase that can bind to this single-stranded promoter andinitiate transcription of RNA therefrom in a 5′-to-3′ direction usingsingle-stranded DNA which is 5′- of and covalently linked to thepromoter. The promoter is oriented within the single-stranded DNA of abipartite target probe 3′- of the target-complementary sequence at the5′-end of the 5′-portion.

[0470] In the embodiment of a method shown in FIG. 24, the gap betweenthe target-complementary sequences of a bipartite target probe annealedto a target sequence is filled by contacting the target sequence towhich a bipartite target probe is annealed with a DNA polymerase underpolymerization conditions. Then, the 5′-phosphorylated end of abipartite target probe annealed to a target sequence is joined to the3′-end of the DNA polymerase-extended 3′-end of said bipartite targetprobe with a ligase, which ligase has little or no activity in ligatingfree ends that are not annealed to a complementary sequence but isactive in joining a 5′-phosphorylated end to an adjacent 3′-hydroxylatedend when the ends are annealed to a complementary DNA sequence,generates a circular transcription substrate. Transcription of thecircular transcription substrate results in synthesis of RNA that iscomplementary to the circular transcription substrate and that can beused to detect the presence of the target sequence.

[0471] Thus, referring to FIG. 24, one embodiment of the presentinvention comprises a method for detecting a target sequence, saidmethod comprising:

[0472] a. providing a bipartite target probe, wherein said bipartitetarget probe comprises a linear single-stranded DNA (ssDNA) comprisingtwo end portions that are complementary to different non-contiguous 5′-and 3′-end portions of a target sequence;

[0473] b. annealing said bipartite target probe to said target nucleicacid comprising said target sequence under hybridization conditions;

[0474] c. contacting said complex comprising said bipartite target probeannealed to said target nucleic acid with a DNA polymerase undernon-strand-displacing DNA polymerization conditions so as to obtain aDNA polymerase extension product that is complementary to the targetsequence between the target-complementary sequences of said annealedbipartite target probe so as to completely fill said gap and so that the3′-end of said synthesized DNA is contiguous with the 5′-end of saidbipartite target probe;

[0475] d. ligating the 5′-end of said bipartite target probe annealed tosaid target nucleic acid with the 3′-end of said DNA polymeraseextension product under ligation conditions with a ligase, wherein saidligase has little or no activity in ligating blunt ends and issubstantially more active in ligating said ends of said simple targetprobes and said ends of said bipartite target probe if said ends areadjacent when annealed to two contiguous regions of a target sequencethan if said ends are not annealed to said target sequence, so as toobtain a circular ssDNA molecule that comprises a circular transcriptionsubstrate;

[0476] e. obtaining said circular transcription substrate, wherein saidsubstrate comprises a sequence that is complementary to said targetsequence;

[0477] f. contacting said circular transcription substrate with an RNApolymerase under transcription conditions so as to synthesize RNA thatis complementary to said circular transcription substrate; and

[0478] g. detecting the synthesis of RNA resulting from transcription ofsaid circular transcription substrate, wherein said synthesis of saidRNA indicates the presence of said target sequence comprising saidtarget nucleic acid.

[0479] In addition to the embodiments disclosed above for filling a gapbetween target-complementary sequences of a bipartite target probe thatare not contiguous when annealed to a target sequence, the inventionalso comprises methods that use a combination of both one or more simpletarget probes and DNA polymerase extension in order to fill the gap soas to obtain adjacent target-complementary sequences prior to theligation step.

[0480] Other embodiments of methods of the invention generate a lineartranscription substrate for amplifying, detecting and quantifying one ormultiple target nucleic acid sequences in a sample, including targetsequences that differ by as little as one nucleotide. FIG. 25 shows abasic embodiment of a method that generates a linear transcriptionsubstrate. This embodiment uses only monopartite target probes. Amonopartite target probe is a single-stranded DNA molecule thatcomprises only one target-complementary sequence, although a monopartitetarget probe can comprise other sequences that are not complementary toa target sequence. By way of example, but not of limitation, a method ofthe invention that generates a linear transcription substrate alwaysuses a monopartite target probe called a “promoter target probe.” A“promoter target probe” has a 5′-portion and a 3′-portion. The5′-portion of a promoter target probe comprises a sequence that iscomplementary to the most 5′-portion of a target sequence, and the3′-portion of a promoter target probe comprises a sequence that servesas a functional transcription promoter for a DNA-dependent RNApolymerase that can bind to this single-stranded promoter and initiatetranscription of RNA therefrom in a 5′-to-3′ direction undertranscription conditions using single-stranded DNA that is 5′- of (withrespect to the same strand) and covalently linked to the promoter as atemplate. The sequence at the 5′-end of the promoter target probepreferably has a 5′-phosphate group or is phosphorylated by apolynucleotide kinase during the course of a method of the invention.The embodiment of the method shown in FIG. 25 also uses anothermonopartite target probe called a “signal target probe.” A “signaltarget probe” has a 3′-portion and a 5′-portion. At least the 3′-endportion of a signal target probe comprises a sequence that iscomplementary to the most 3′-portion of a target sequence. As shown inthe embodiment in FIG. 25, the 3′-end of the signal target probe has a3′-hydroxyl group. The 5′-portion of a signal target probe comprises a“signal sequence.” A signal sequence is a sequence that is detectable insome way following its transcription during a method of the invention.The invention does not require the use of a signal target probe having asignal sequence. By way of example, but not of limitation, a simpletarget probe could be used in an assay of the invention in place of asignal target probe. If a signal target probe is used in a method of theinvention, the signal sequence can comprise any sequence that isdetectable following transcription. By way of example, but not oflimitation, a signal sequence can comprise a sequence that is detectableusing a molecular beacon as described by Tyagi et al. (U.S. Pat. Nos.5,925,517 and 6,103,476 of Tyagi et al. and 6,461,817 of Alland et al.,all of which are incorporated herein by reference). A preferred signalsequence of the invention is a sequence that results in an additionalamplification of the signal following its transcription, thus making thedetection of a target sequence more sensitive. The signal target probeused in the method shown in FIG. 25 can be, for example, a signalsequence that encodes a substrate for Q-beta replicase (EPICENTRETechnologies, Madison, Wis.), which permits additional amplification ofthe signal by incubating the transcription product with Q-beta replicaseunder replication conditions. However, as discussed elsewhere herein,many other signal sequences can be used in a signal target probe, all ofwhich are incorporated as part of the present invention.

[0481] Thus, again referring to FIG. 25, one embodiment of the presentinvention comprises a method for detecting a target, said methodcomprising:

[0482] a. providing a promoter target probe, wherein said promotertarget probe comprises a linear single-stranded DNA (ssDNA) comprising a5′-end portion that is complementary to the most 5′-portion of saidtarget sequence and, 3′- of said target-complementary portion, atranscription promoter for an RNA polymerase that can bind to saidsingle-stranded promoter and initiate transcription of RNA therefrom ina 5′-to-3′ direction using single-stranded DNA that is 5′- of andcovalently linked to said promoter as a template;

[0483] b. providing a signal target probe, wherein said signal targetprobe comprises a linear ssDNA comprising a 3′-end portion that iscomplementary to the most 3′-portion of said target sequence and a5′-portion comprising a signal sequence;

[0484] c. optionally, provided said target-complementary sequences ofsaid promoter target probe and said signal target probe are notcontiguous when annealed to said target sequence, providing one or moresimple target probes, wherein said simple target probes arecomplementary to said target sequence so as to anneal to said targetsequence in the gap between the target-complementary sequences of saidpromoter target probe and said signal target probe so as to completelyfill said gap and so that each of the ends of said simple target probesare contiguous with an end of a simple target probe or with a 5′-end ofsaid promoter target probe or a 3′-end of said signal target probe;

[0485] d. annealing said promoter target probe, said simple targetprobes, if present, and said signal target probe to said target nucleicacid comprising said target sequence under hybridization conditions;

[0486] e. ligating said promoter target probe, said simple targetprobes, if present, and said signal target probe that are annealed tosaid target nucleic acid under ligation conditions with a ligase,wherein said ligase has little or no activity in ligating blunt ends andis substantially more active in ligating said ends of said target probesif said ends are adjacent when annealed to two contiguous regions of atarget sequence than if said ends are not annealed to said targetsequence, so as to obtain a linear ssDNA molecule that comprises alinear transcription substrate;

[0487] f. obtaining said linear transcription substrate, wherein saidlinear transcription substrate comprises a sequence that iscomplementary to said target sequence;

[0488] g. contacting said linear transcription substrate with an RNApolymerase under transcription conditions so as to synthesize RNA thatis complementary to said target-complementary sequence and said signalsequence of said linear transcription substrate; and

[0489] h. detecting the synthesis of RNA resulting from transcription ofsaid linear transcription substrate, wherein said synthesis of said RNAindicates the presence of said target sequence comprising said targetnucleic acid.

[0490] In addition to the embodiment shown in FIG. 25 in which a simpletarget probe is used to fill a gap on a target sequence between thetarget-complementary sequences of a promoter target probe and a signaltarget probe, the invention also comprises embodiments in which a DNApolymerase is used to fill the gap between a promoter target probe and asignal target probe, wherein said target probes are not adjacent whenannealed to a target sequence. The invention further comprises use of acombination of a promoter target probe, one or more simple targetprobes, a signal target probe and DNA polymerase extension of the3′-hydroxyl end of said signal target probe or of one or more simpletarget probes so that said target probes, including said DNApolymerase-extended target probes, completely fill the gap on a targetsequence between the target-complementary portions of said promoterprobe and said signal target probe. Thus any combination of simpletarget probes and DNA polymerase extension can be used to fill the gapbetween the target-complementary portions of the promoter probe and thesignal target probe so as to obtain adjacent target-complementarysequences prior to the ligation step.

[0491] The invention also comprises methods for obtaining secondary oradditional amplification by using the RNA products synthesized bytranscription of a circular transcription substrate or a lineartranscription substrate as a template for ligation of the same ordifferent bipartite or monopartite target probes, thus generatingadditional circular transcription substrates or linear transcriptionsubstrates, respectively. By way of example, but not of limitation, oneembodiment of a method of the invention for obtaining secondaryamplification uses bipartite target probes and two ligases—one ligasethat can ligate target-complementary sequences of a bipartite targetprobe annealed to a DNA target sequence to form a first circulartranscription substrate, and one that can ligate the sametarget-complementary sequences of said bipartite target probe annealedto an RNA transcript resulting from transcription of said first circulartranscription substrate. By way of example, but not of limitation, oneligase that can be used in a method of the present invention forligation of contiguous DNA molecules annealed to an RNA ligationtemplate is T4 RNA ligase (Epicentre Technologies, Madison, Wis., USA),as disclosed by Faruqi in U.S. Pat. No. 6,368,801 B1. The invention alsocomprises embodiments that use similar secondary amplification methodswith two ligases using monopartite target probes and that generatelinear transcription substrates.

[0492] In addition to comprising embodiments of methods whereinbipartite target probes are used that anneal to both the target sequenceand to the same sequence in the RNA transcripts resulting fromtranscription of a first circular transcription substrate, the inventionalso comprises other embodiments of methods and assays wherein a secondbipartite target probe is used that anneals to a sequence in the RNAtranscript that is complementary to a signal sequence or an optionalsequence of the first circular transcription substrate rather thanannealing to the target sequence or the identical sequence in the RNAtranscript.

[0493] In still other embodiments, the invention also comprises use of areverse transcriptase process to obtain additional amplification of atarget sequence and/or a signal sequence in an assay or method of theinvention. An example of an embodiment of the invention that uses areverse transcriptase process is shown in FIG. 26. This exampleillustrates a number of aspects of the invention that result inimprovements over the methods and assays of the prior art.

[0494] The first part of the assay or method in FIG. 26 is similar tothe embodiment shown in FIG. 21. Thus, a first circular transcriptionsubstrate is generated by ligation of a first bipartite target probeannealed to a target sequence in a sample. Then, in vitro transcriptionof the first circular transcription substrate amplifies the targetsequence and the signal sequence, if present. In the example, shown inFIG. 26, rolling circle transcription is used to synthesize RNAcomprising multimeric copies of an RNA oligomer that is complementary tothe first circular transcription substrate. In contrast to run-offtranscription of linear transcription substrates, as is used for methodsin the prior art such as, but not limited to, NASBA or TMA, rollingcircle transcription synthesizes RNA tht has sequences that arecomplementary to the single-stranded transcription promoter in acircular transcription substrate. As discussed below, the presence ofthese promoter-complementary sequences in the RNA transcription productfrom rolling circle transcription permits generation of additionalsingle-stranded transcription promoters that can initiate additional invitro transcription reactions and thereby further amplify the targetsequence and/or signal sequence.

[0495] Thus, one or more oligonucleotide primers anneal to themultimeric RNA transcription products and first-strand cDNA issynthesized by extension of said primers by a reverse transcriptaseunder reverse transcription reaction conditions. In the example shown inFIG. 26, only one reverse transcription primer is used that anneals tothe same sequence in different repeated sites on the multimeric RNA.However, the invention also comprises embodiments that use multiplereverse transcription primers, each of which is complementary to adifferent sequence of an RNA oligomer that is, in turn, complementary toa circular transcription substrate. The sequence to which a reversetranscription primer anneals in an RNA multimer can also vary.Preferably, a reverse transcription primer anneals to a sequence in theRNA multimer in a region that is complementary to an optional sequenceportion of a circular transcription substrate and that is 3′- of asignal sequence-complementary sequence, if present. The reversetranscription primer shown in FIG. 26 anneals to the RNA multimer at asite that is 3′ of of a signal sequence-complementary sequence of eacholigomer of the multimer. The reverse transcription primer shown in FIG.26 has a 5′-portion comprising a “tail” that is a sequence that is notcomplementary to the RNA transcript. The use of a tail is optional andis not required for methods and assays of the invention.

[0496] Again referring to FIG. 26, following reverse transcription ofthe RNA multimer, the first-strand cDNA is available in the reactionmixture for at least two subsequent functions. First, the first-strandcDNA has a functional single-stranded transcription promoter and is usedas a linear transcription substrate for synthesis of RNA using the RNApolymerase that initiates transcription from said promoter undertranscription conditions. Synthesis of RNA corresponding to the targetsequence and/or the signal sequence in these linear transcriptionsubstrates can be detected according to the detection method used in theparticular embodiment of an assay or method of the invention. Second,the first strand cDNA can be used as a ligation template for ligation ofa second bipartite target probe under ligation conditions. In theembodiment shown in FIG. 26, the second bipartite target probe isidentical to the first bipartite target probe except that with respectto the target-complementary sequences at the 3′- and 5′-ends of saidsecond bipartite target probe. The 5′-end portion of the secondbipartite target probe comprises a sequence that is complementary to thetarget-complementary sequence at the 3′-end portion of the firstbipartite target probe, and this sequence is in turn covalently attachedand 5′- of a promoter sequence in the 5′-portion of the second bipartitetarget probe. The 3′-end portion of the second bipartite target probecomprises a sequence that is complementary to the target-complementarysequence at the 5′-end portion of the first bipartite target probe, andthis sequence is in turn covalently attached and 3′- of a signalsequence in the 3′-portion of the second bipartite target probe, if asignal sequence is present. Thus, the sequences at the 3′- and 5′-endsof said second bipartite target probe are identical to the targetsequence and are complementary to the target-complementary sequences inboth the first circular transcription substrate and in the first-strandcDNA obtained by reverse transcription of RNA transcripts from saidfirst circular transcription substrate, both of which thus serve asligation templates for ligation of the second bipartite target probe bya ligase under ligation conditions. Ligation of a second bipartitetarget probe generates a second circular transcription substrate.

[0497] The second circular transcription substrate is then a substratefor rolling circle transcription, generating a complementary RNAmultimer transcript. The RNA multimer transcript resulting from rollingcircle transcription is then a substrate for reverse transcription by areverse transcriptase under reverse transcription conditions. Since, inthe embodiment shown in FIG. 26, the second circular transcriptionsubstrate is identical to the first circular transcription substrate inall portions except for the target-complementary portion, the samereverse transcription primer can be used to generate first-strand cDNAthat is complementary to the RNA multimer from the second circulartranscription substrate. The resulting first-strand cDNA is a secondlinear transcription substrate. In vitro transcription of said secondlinear transcription substrate by an RNA polymerase that initiatestranscription using said single-stranded transcription promoter undertranscription conditions generates RNA transcripts that can be detectedin the assay or method. The sequence corresponding to a target sequencein said first-strand cDNA also serves as a template for ligation of afirst bipartite target probe by a ligase under ligation conditions.Ligation of another first bipartite target probe forms another firstcircular transcription substrate. Thus, the various annealing, ligation,rolling circle transcription, reverse transcription, and linear run-offtranscription processes of this embodiment of an assay or method of theinvention can continue, with continual generation of RNA that can bedetected according to the particular assay or method until one or moreof the reaction components are exhausted. The repeating cycles ofprocesses of this embodiment of an assay or method results in highsensitivity and shorter reaction times, while retaining a high degree ofspecificity.

[0498] Thus, again referring to FIG. 26, one embodiment of the presentinvention comprises a method for detecting a target sequence, saidmethod comprising:

[0499] a. providing a first bipartite target probe, wherein said firstbipartite target probe comprises a 5′-portion and a 3′-portion, whereinsaid 5′-portion comprises: (i) a 5′-end portion that comprises asequence that is complementary to a target sequence, and (ii) a promotersequence, wherein said promoter sequence is covalently attached to and3′- of said target-complementary sequence in said 5′-portion; andwherein said 3′-portion comprises: (i) a 3′-end portion that comprises asequence that is complementary to a target sequence, wherein saidtarget-complementary sequence of said 3′-end portion, when annealed tosaid target sequence, is adjacent to said target-complementary sequenceof said 5′-end portion of said first bipartite target probe, and (ii)optionally, a signal sequence, wherein said signal sequence is 5′- ofsaid target-complementary sequence of said 3′-portion of said firstbipartite target probe;

[0500] b. providing a second bipartite target probe, wherein said secondbipartite target probe comprises a 5′-portion and a 3′-portion, whereinsaid 5′-portion comprises: (i) a 5′-end portion that comprises sequencethat is complementary to said target-complementary sequence of said3′-end portion of said first bipartite target probe, and (ii) a promotersequence, wherein said promoter sequence in said 5′-portion of saidsecond bipartite target probe is 3′- of said target-complementarysequence in said 5′-portion; and wherein said 3′-portion comprises: (i)a 3′-end portion that comprises sequence that is complementary to saidtarget-complementary sequence of said 5′-end portion of said firstbipartite target probe, and (ii) optionally, a signal sequence, whereinsaid signal sequence in said 3′-portion of said second bipartite targetprobe is 5′- of said target-complementary sequence in said 3′-portion;

[0501] c. annealing said first bipartite target probe to said targetnucleic acid comprising said target sequence under hybridizationconditions;

[0502] d. ligating said first bipartite target probe annealed to saidtarget nucleic acid under ligation conditions with a ligase, whereinsaid ligase has little or no activity in ligating blunt ends and issubstantially more active in ligating said ends of said first bipartitetarget probe if said ends are adjacent when annealed to two contiguousregions of a target sequence than if said ends are not annealed to saidtarget sequence, so as to obtain a circular ssDNA molecule thatcomprises a first circular transcription substrate;

[0503] e. obtaining said first circular transcription substrate;

[0504] f. contacting said first circular transcription substrate with anRNA polymerase under transcription conditions so as to synthesize RNAthat is complementary to said first circular transcription substrate;

[0505] g. annealing to said RNA that is complementary to said firstcircular transcription substrate a primer, wherein said primer iscomplementary to said RNA;

[0506] h. contacting said RNA to which said primer is annealed with areverse transcriptase under reverse transcription conditions so as toobtain a first first-strand cDNA;

[0507] i. obtaining said first first-strand cDNA;

[0508] j. annealing to said first first-strand cDNA said secondbipartite target probe under annealing conditions;

[0509] k. contacting said first first-strand cDNA to which said secondbipartite target probe is annealed with a a ligase, wherein said ligasehas little or no activity in ligating blunt ends and is substantiallymore active in ligating said ends of said second bipartite target probeif said ends are adjacent when annealed to two contiguous regions ofsaid first first-strand cDNA than if said ends are not annealed to saidsequence, so as to obtain a circular ssDNA molecule that comprises asecond circular transcription substrate;

[0510] l. obtaining said second circular transcription substrate;

[0511] m. contacting said second circular transcription substrate withan RNA polymerase under transcription conditions so as to synthesize RNAthat is complementary to said second circular transcription substrate;

[0512] n. annealing to said RNA that is complementary to said secondcircular transcription substrate a primer, wherein said primer iscomplementary to said RNA;

[0513] o. contacting said RNA to which said primer is annealed with areverse transcriptase under reverse transcription conditions so as toobtain a second first-strand cDNA;

[0514] p. obtaining said second first-strand cDNA;

[0515] q. annealing to said second first-strand cDNA said firstbipartite target probe under annealing conditions;

[0516] r. contacting said second first-strand cDNA to which said firstbipartite target probe is annealed with a a ligase, wherein said ligasehas little or no activity in ligating blunt ends and is substantiallymore active in ligating said ends of said first bipartite target probeif said ends are adjacent when annealed to two contiguous regions ofsaid second first-strand cDNA than if said ends are not annealed to saidsequence, so as to obtain a circular ssDNA molecule that comprises athird circular transcription substrate that is identical to said firstcircular transcription substrate;

[0517] s. obtaining said third circular transcription substrate that isidentical to said first circular transcription substrate;

[0518] t. repeating steps a through t; and

[0519] u. detecting the synthesis of RNA resulting from transcription ofsaid first, second and third circular transcription substrates and fromsaid first and second linear transcription substrates, wherein saidsynthesis of said RNA indicates the presence of said target sequencecomprising said target nucleic acid.

[0520] The methods and assays of the embodiment of the invention shownin FIG. 26 can be performed in a stepwise manner or, more preferably, ina single reaction mixture in a continuous manner. Thus, one embodimentof the present invention comprises a method for detecting a targetsequence, said method comprising:

[0521] l. providing a reaction mixture comprising:

[0522] a. a first bipartite target probe, wherein said first bipartitetarget probe comprises a 5′-portion and a 3′-portion, wherein said5′-portion comprises: (i) a 5′-end portion that comprises a 5′-phosphategroup and a sequence that is complementary to a target sequence, and(ii) a promoter sequence, wherein said promoter sequence is covalentlyattached to and 3′- of said target-complementary sequence in said5′-portion; and wherein said 3′-portion comprises: (i) a 3′-end portionthat comprises a sequence that is complementary to a target sequence,wherein said target-complementary sequence of said 3′-end portion, whenannealed to said target sequence, is adjacent to saidtarget-complementary sequence of said 5′-end portion of said firstbipartite target probe, and (ii) optionally, a signal sequence, whereinsaid signal sequence is 5′- of said target-complementary sequence ofsaid 3′-portion of said first bipartite target probe;

[0523] b. a second bipartite target probe, wherein said second bipartitetarget probe comprises a 5′-portion and a 3′-portion, wherein said5′-portion comprises: (i) a 5′-end portion that comprises a 5′-phosphategroup and sequence that is complementary to said target-complementarysequence of said 3′-end portion of said first bipartite target probe,and (ii) a promoter sequence, wherein said promoter sequence in said5′-portion of said second bipartite target probe is 3′- of saidtarget-complementary sequence in said 5′-portion; and wherein said3′-portion comprises: (i) a 3′-end portion that comprises sequence thatis complementary to said target-complementary sequence of said 5′-endportion of said first bipartite target probe, and (ii) optionally, asignal sequence, wherein said signal sequence in said 3′-portion of saidsecond bipartite target probe is 5′- of said target-complementarysequence in said 3′-portion;

[0524] c. a ligase, wherein said ligase has little or no activity inligating blunt ends and is substantially more active in ligating theends of a bipartite target probe if said ends are adjacent when annealedto two contiguous regions of a complementary sequence than if said endsare not annealed to said complementary sequence, so as to obtain acircular ssDNA molecule that comprises a circular transcriptionsubstrate;

[0525] d. a reverse transcriptase and one or more primers, wherein atleast the 3′-portion of one said primer comprises a sequence that iscomplementary to a sequence of said first bipartite target probe and ofsaid second bipartite target probe and wherein said complementaryportion of said primer is not complementary to said target sequence orthe complement of said target sequence;

[0526] e. an RNA polymerase, wherein said RNA polymerase recognizes saidsingle-stranded transcription promoters of said first and secondbipartite target probes and synthesizes RNA therefrom using as atemplate single-stranded DNA to which said promoters are functionallyattached;

[0527] f. optionally, a single strand binding protein;

[0528] g. optionally, a detection oligo, wherein said detection oligoanneals to an RNA transcript sequence that is complementary to a signalsequence of said first and/or second bipartite target probe; and

[0529] h. reaction conditions wherein said ligase, said reversetranscriptase, and said RNA polymerase are optimally active incombination and wherein said target-complementary sequences of saidfirst bipartite target probe anneal to said target sequence, if present,with specificity, and;

[0530] 2. contacting said reaction mixture from step 1 above with asample comprising a target nucleic acid comprising a target sequence, ifpresent, wherein said reaction mixture containing said sample ismaintained at a temperature wherein said ligase, said reversetranscriptase, and said RNA polymerase are optimally active incombination and wherein said target-complementary sequences of saidfirst bipartite target probe anneal to said target sequence, if present,with specificity, and wherein said temperature of said reaction mixtureis maintained for a time sufficient to permit synthesis of RNAtranscription products complementary to circular transcriptionsubstrates obtained from said bipartite target probes if said targetsequence is present in said sample; and

[0531] 3. detecting the synthesis of RNA resulting from transcription ofsaid circular transcription substrates, wherein said synthesis of saidRNA indicates the presence of said target sequence comprising saidtarget nucleic acid.

[0532] In preferred embodiments of the above methods or assays fordetecting a target sequence, said ligase comprises a ligase chosen fromamong Ampligases thermostable DNA Ligase, Tth DNA Ligase, Tfl DNALigase, Tsc DNA Ligase, or Pfu DNA Ligase.

[0533] In preferred embodiments of the above methods or assays fordetecting a target sequence, said reverse transcriptase is a reversetranscriptase that has RNase H activity, wherein said reversetranscriptase is chosen from among MMLV reverse transcriptase, AMVreverse transcriptase, another retroviral reverse transcriptase, or areverse transcriptase encoded by a thermostable phage. In otherembodiments of the above methods or assays, said reverse transcriptasecomprises a DNA polymerase chosen from among IsoTherm™ DNA polymerase,Bst DNA polymerase large fragment, BcaBEST™ DNA polymerase, and Tth DNApolymerase.

[0534] In preferred embodiments of the above methods or assays fordetecting a target sequence, said RNA polymerase is N4 mini-vRNAP.

[0535] R. Methods and Assays for Detecting and Quantifying Non-NucleicAcid Analytes Using Target Sequence Tags Comprising or Attached toAnalyte-Binding Substances

[0536] The present invention includes methods, compositions and kitsthat use an analyte-binding substance for detecting an analyte in asample. An “analyte-binding substance” is a substance that binds ananalyte that one desires to detect in an assay or method of theinvention. An analyte-binding substance is also referred to as an“affinity molecule,” an “affinity substance,” a “specific bindingsubstance,” or a “binding molecule” for an analyte. Usually, an analytemolecule and an analyte-binding substance or affinity molecule for theanalyte molecule are related as a specific “binding pair”, i.e., theirinteraction is only through non-covalent bonds such as hydrogen-bonding,hydrophobic interactions (including stacking of aromatic molecules), vander Waals forces, and salt bridges. Without being bound by theory, it isbelieved in the art that these kinds of non-covalent bonds result inbinding, in part due to complementary shapes or structures of themolecules involved in the binding pair.

[0537] The term “binding” according to the invention refers to theinteraction between an analyte-binding substance or affinity moleculeand an analyte as a result of non-covalent bonds, such as, but notlimited to, hydrogen bonds, hydrophobic interactions, van der Waalsbonds, and ionic bonds.

[0538] In most embodiments of the invention, target probes are used todetect an analyte comprising a target sequence in a target nucleic acid.Following annealing and joining of target probes in the presence of atarget sequence in a sample, the resulting transcription substrate isamplified by transcription using an RNA polymerase, and the presence ofan RNA complementary to the transcription substrate indicates that thetarget sequence was present in the sample.

[0539] However, a nucleic acid can also be used in a method of thepresent invention as an analyte-binding substance to detect an analytethat does not comprise a nucleic acid. By way of example, but not oflimitation, a method termed “SELEX,” as described by Gold and Tuerk inU.S. Pat. No. 5,270,163, which is incorporated herein by reference, canbe used to select a nucleic acid for use as an analyte-binding substancein a method of the invention for detecting an analyte comprising almostany molecule in a sample. SELEX permits selection of a nucleic acidmolecule that has high affinity for a specific analyte from a largepopulation of nucleic acid molecules, at least a portion of which have arandomized sequence. For example, a population of all possiblerandomized 25-mer oligonucleotides (i.e., having each of four possiblenucleic acid bases at every position) will contain 425 (or 1015)different nucleic acid molecules, each of which has a differentthree-dimensional structure and different analyte binding properties.SELEX can be used, according to the methods described in U.S. Pat. Nos.5,270,163; 5,567,588; 5,580,737; 5,587,468; 5,683,867; 5,696,249;5723,594; 5,773,598; 5,817,785; 5,861,254; 5,958,691; 5,998,142;6,001,577; 6,013,443; and 6,030,776, all of which are incorporatedherein by reference, in order to select an analyte-binding nucleic acidwith high affinity for a specific analyte that is not a nucleic acid orpolynucleotide for use in a method or assay of the invention. Onceselected using SELEX, analyte-binding substances or affinity moleculescomprising nucleic acid molecules can be made for use in the methods ofthe present invention by using any of numerous in vivo or in vitrotechniques known in the art, including, by way of example, but not oflimitation, automated nucleic acid synthesis techniques, PCR, or invitro transcription. A nucleic acid molecule that is an analyte-bindingsubstance that has been selected using SELEX can be detected usingbipartite or monopartite target probes in a similar way to how suchtarget probes are used to detect a target sequence in a target nucleicacid analyte, as described elsewhere herein. Since an analyte-bindingsubstance that is selected using SELEX comprises a nucleic acid, acontinuous sequence within the analyte-binding substance can be used asa “target sequence” and target probes can be designed, wherein thetarget-complementary sequences in said target probes are complementaryto said continuous sequence in said analyte-binding substance. Anotherimportant aspect of these embodiments of the invention is that saidtarget sequence in said analyte-binding substance that was selectedusing SELEX should be capable of annealing to said target probes whensaid analyte-binding substance is also bound to an analyte; i.e., thebinding to the analyte does not block annealing of target probes to thetarget sequence.

[0540] Thus, another embodiment of the present invention is a method fordetecting an analyte in a sample, wherein said analyte comprises abiomolecule that is not a nucleic acid, said method comprising:

[0541] a. providing an analyte-binding substance comprising a nucleicacid, wherein said nucleic acid binds with selectivity and high affinityto said analyte;

[0542] b. providing target probes comprising either (i) a promotertarget probe and one or more additional target probes chosen from amonga signal target probe and simple target probe; or (ii) a bipartitetarget probe and, if said target-complementary sequences of saidbipartite target probe are not contiguous when annealed to said targetsequence in said analyte-binding substance, optionally, one or moresimple target probes; wherein said target probes of (i) or (ii) comprisesequences that are complementary to adjacent regions of a targetsequence in said analyte-binding substance;

[0543] c. contacting said analyte-binding substance to an analyte in asample;

[0544] d. separating said analyte-binding substance molecules that arebound to said analyte from said analyte-binding substance molecules thatare not bound to said analyte;

[0545] e. contacting said analyte-binding substance molecules that arebound to said analyte with said target probes provided in step b(i) orstep b(ii) above under hybridization conditions that permit said targetprobes that are complementary to said target sequences in saidanalyte-binding substance to anneal thereto;

[0546] f. ligating said adjacent target probes that are annealed to saidtarget sequence of said analyte-binding substance with a ligase underligation conditions so as to obtain a transcription substrate;

[0547] g. obtaining said transcription substrate;

[0548] h. contacting said transcription substrate with an RNA polymeraseunder transcription conditions so as to synthesize RNA that iscomplementary to said transcription substrate;

[0549] i. optionally, repeating steps a through i; and

[0550] j. detecting the synthesis of RNA resulting from transcription ofsaid transcription substrate, wherein said synthesis of said RNAindicates the presence of said analyte in said sample.

[0551] Thus, the use of an analyte-binding substance comprising anucleic acid selected using SELEX permits the methods of the presentinvention to be used to detect other analyte molecules that are notnucleic acids.

[0552] The nucleic acid molecules that contain a randomized sequencethat are used to generate a library of molecules for selection of ananalyte-binding substance using SELEX can also be made using methodssimilar to those described by Ohmichi et al. (Proc. Natl. Acad. Sci.USA, 99: 54-59, 2002), incorporated herein by reference. Thus, randomsequence circular DNA molecules comprising about 103 nucleotides, ofwhich about 40 nucleotides comprise randomized sequence are repeatedlyselected for binding to an analyte by: binding the circular DNAs to ananalyte attached to a surface; washing away the unbound circular DNAmolecules; recovering the circular DNAs bound to the analyte; obtainingRNA complementary to the recovered circular DNA molecules by rollingcircle transcription; amplifying the RNA by RT-PCR using one5′-biotinylated primer; immobilizing the RT-PCR product on a surfacewith streptavidin; obtaining the strand of the RT-PCR product that doesnot contain biotin; and then ligating the single-stranded RT-PCR strand(using a ligation splint) to obtain the first round of selected circularDNA molecules. The first round of circular DNA molecules is then boundto an analyte as just described, and the whole process is again repeatedfor a total of about 15 rounds of selection of circular DNA moleculesfor analyte binding. The selected circular DNA molecules are thenanalyzed for analyte binding in order to obtain an analyte-bindingsubstance for use in an assay or method of the present invention. Asdescribed above related to SELEX, a target sequence in the selectedanalyte-binding substance can be detected using monopartite or bipartitetarget probes as described elsewhere herein. Thus, an analyte-bindingsubstance is used to bind an analyte in a sample and then, afterremoving unbound analyte-binding substance (if the analyte-bindingsubstance is attached to a surface or becomes attached to a surfaceduring a process of the assay or method), the analyte-binding substanceis detected using target probes that are complementary to a targetsequence in the analyte-binding substance. A method for detecting ananalyte-binding substance can comprise a step comprising ligation oftarget probes of the invention as described in the embodiments of themethod immediately above herein for detecting a target sequence in ananalyte-binding substance that is bound to an analyte. However, in someother embodiments for detecting an analyte-binding substance that isbound to an analyte, a ligation step is omitted and the analyte-bindingsubstance:analyte complex is detected by annealing to said complex atranscription substrate that contains a sequence that is complementaryto a target sequence in said analyte-binding substance. After removingunhybridized transcription substrates, transcription substrates that areannealed to said analyte-binding substance: analyte complex are detectedby synthesis of RNA resulting from in vitro transcription of thecomplex-bound transcription substrate.

[0553] A “peptide nucleic acid (PNA)” or a molecule comprising both anucleic acid and a PNA, as described in U.S. Pat. Nos. 5,539,082;5,641,625; 5,700,922; 5,705,333; 5,714,331; 5,719,262; 5,736,336;5,773,571; 5,786,461; 5,817,811; 5,977,296; 5,986,053; 6,015,887; and6,020,126, which are incorporated herein by reference, can also be usedin methods of the present invention as an analyte-binding substance fora non-nucleic acid analyte. In general, a PNA molecule is a nucleic acidanalog consisting of a backbone comprising, for example,N-(2-aminoethyl)glycine units, to each of which a nucleic acid base islinked through a suitable linker, such as, but not limited to an aza,amido, ureido, or methylene carbonyl linker. The nucleic acid bases inPNA molecules bind complementary single-stranded DNA or RNA according toWatson-Crick base-pairing rules. However, the T_(m)′s for PNA/DNA orPNA/RNA duplexes or hybrids are higher than the T_(m)′s for DNA/DNA,DNA/RNA, or RNA/RNA duplexes. In these embodiments, a “PNA targetsequence” is present in said analyte-binding substance comprising PNA,to which, target-complementary sequences of monopartite or bipartitetarget probes (or transcription substrates) can anneal, permittingdetection as described above for analyte-binding molecules selectedusing SELEX. Thus, PNA used as an analyte-binding substance in an assayor method of the present invention provides tighter binding (and greaterbinding stability) for target-complementary sequences in target probesor transcripton substrates (e.g., see U.S. Pat. No. 5,985,563). Also,since PNA is not naturally occurring, PNA molecules are highly resistantto protease and nuclease activity. PNA for use as an analyte bindingsubstance can be prepared according to methods known in the art, suchas, but not limited to, methods described in the above-mentionedpatents, and references therein. Antibodies to PNA/analyte complexes canbe used in the invention for capture, recognition, detection,identification, or quantitation of nucleic acids in biological samples,via their ability to bind specifically to the respective complexeswithout binding the individual molecules (U.S. Pat. No. 5,612,458).

[0554] The invention also contemplates that a combinatorial library ofrandomized peptide nucleic acids prepared by a method such as, but notlimited to, the methods described in U.S. Pat. Nos. 5,539,083;5,831,014; and 5,864,010, can be used to prepare analyte-bindingsubstances for use in assays for analytes of all types, includinganalytes that are nucleic acids, proteins, or other analytes, withoutlimit. As is the case for the SELEX method with nucleic acids,randomized peptide or peptide nucleic acid libraries are made to containmolecules with a very large number of different binding affinities foran analyte. After selection of an appropriate affinity molecule for ananalyte from a library, the selected affinity molecule can be used inthe invention as an analyte-binding substance in the second portion ofthe reporter probe.

[0555] An analyte-binding substance can also be an oligonucleotide orpolynucleotide with a modified backbone that is not an amino acid, suchas, but not limited to modified oligonucleotides described in U.S. Pat.Nos. 5,602,240; 6,610,289; 5,696,253; or 6,013,785.

[0556] The invention also contemplates that an analyte-binding substancecan be prepared from a combinatorial library of randomized peptides(i.e., comprising at least four naturally-occurring amino acids). Oneway to prepare the randomized peptide library is to place a randomizedDNA sequence, prepared as for SELEX, downstream of a phage T7 RNApolymerase promoter, or a similar promoter, and then use a method suchas, but not limited to, coupled transcription-translation, as describedin U.S. Pat. Nos. 5,324,637; 5,492,817; or 5,665,563, or stepwisetranscription, followed by translation. Alternatively, a randomized DNAsequence, prepared as for SELEX, can be cloned into a site in a DNAvector that, once inserted, encodes a recombinant MDV-1 RNA containingthe randomized sequence that is replicatable by Q.beta. replicase (e.g.,between nucleotides 63 and 64 in MDV-1 (+) RNA; see U.S. Pat. No.5,620,870). The recombinant MDV-1 DNA containing the randomized DNAsequence is downstream from a T7 RNA polymerase promoter or a similarpromoter in the DNA vector. Then, following transcription, therecombinant MDV-1 RNA, containing the randomized sequence can be used tomake a randomized peptide library comprising at least four naturallyoccurring amino acids by coupled replication-translation as described inU.S. Pat. No. 5,556,769. An analyte-binding substance can be selectedfrom the library by binding peptides in the library to an analyte,separating the unbound peptides, and identifying one or more peptidesthat is bound to analyte by means known in the art. Alternatively, highthroughput screening methods can be used to screen all individualpeptides in the library to identify those that can be used asanalyte-binding substances. Although the identification of ananalyte-binding peptide by these methods is difficult and tedious, themethods in the art are improving for doing so, and the expenditure oftime and effort required may be warranted for identifyinganalyte-binding substances for use in assays of the invention that willbe used routinely in large numbers.

[0557] In embodiments of the present invention in which ananalyte-binding substance comprises a peptide, a protein, including, butnot limited to an antibody, streptavidin, or another biomolecule, anucleic acid sequence can be attached to said analyte-binding substance,wherein said nucleic acid serves as a “tag” comprising a target sequencethat can be detected using target probes or transcription substrates ofthe invention. In this way, the methods and assays of the invention canbe used for sensitive and specific detection of analytes that are notnucleic acids.

[0558] Analyte-binding substances for particular analytes and methods ofpreparing them are well known in the art. Naturally occurring nucleicacid or polynucleotide sequences that have affinity for other naturallyoccurring molecules such as, but not limited to, protein molecules, areknown in the art, and nucleic acid molecules comprising these sequencescan be used, both as analyte-binding substances and as tags comprisingtarget sequences for detection using target probes or transcriptionsubstrates of the invention. Examples include, but are not limited tocertain nucleic acid sequences such as operators, promoters, origins ofreplication, sequences recognized by steroid hormone-receptor complexes,restriction endonuclease recognition sequences, ribosomal nucleic acids,and so on, which are known to bind tightly to certain proteins. Forexample, in two well-known systems, the lac repressor and thebacteriophage lambda repressor each bind to their respective specificnucleic acid sequences called “operators” to block initiation oftranscription of their corresponding mRNA molecules. Nucleic acidscontaining such specific sequences can be used in the invention asanalyte-binding substances for the respective proteins or othermolecules for which the nucleic acid has affinity. In these cases, thenucleic acid with the specific sequence is used as the analyte-bindingsubstance in assays for the respective specific protein, glycoprotein,lipoprotein, small molecule or other analyte that it binds. One ofseveral techniques that is generally called “footprinting” (e.g., seeGalas, D. and Schmitz, A, Nucleic Acids Res., 5: 3161, 1978) can be usedto identify sequences of nucleic acids that bind to a protein. Othermethods are also known to those with skill in the art and can be used toidentify nucleic acid sequences for use as specific analyte-bindingsubstances for use in the invention.

[0559] A variety of other analyte-binding substances can also be used.For an antigen analyte (which itself may be an antibody), antibodies,including monoclonal antibodies, are available as analyte-bindingsubstances. For certain antibody analytes in samples which include onlyone antibody, an antibody binding protein such as Staphylococcus aureusProtein A can be employed as an analyte-binding substance. For ananalyte, such as a glycoprotein or class of glycoproteins, or apolysaccharide or class of polysaccharides, which is distinguished fromother substances in a sample by having a carbohydrate moiety that isbound specifically by a lectin, a suitable analyte-binding substance isthe lectin. For an analyte that is a hormone, a receptor for the hormonecan be employed as an analyte-binding substance. Conversely, for ananalyte that is a receptor for a hormone, the hormone can be employed asthe analyte-binding substance. For an analyte that is an enzyme, aninhibitor of the enzyme can be employed as an analyte-binding substance.For an analyte that is an inhibitor of an enzyme, the enzyme can beemployed as the analyte-binding substance.

[0560] Based on the definition for “binding,” and the wide variety ofaffinity molecules and analytes that can be used in the invention, it isclear that “binding conditions” vary for different specific bindingpairs. Those skilled in the art can easily determine conditions whereby,in a sample, binding occurs between affinity molecule and analyte thatmay be present. In particular, those skilled in the art can easilydetermine conditions whereby binding between affinity molecule andanalyte that would be considered in the art to be “specific binding” canbe made to occur. As understood in the art, such specificity is usuallydue to the higher affinity of affinity molecule for analyte than forother substances and components (e.g., vessel walls, solid supports) ina sample. In certain cases, the specificity might also involve, or mightbe due to, a significantly more rapid association of affinity moleculewith analyte than with other substances and components in a sample.

[0561] In general, any of the methods and assays described herein todetect and quantify an analyte comprising a target sequence in a targetnucleic acid can also be used to detect and quantify a target sequencethat comprises a target sequence tag that is attached to ananalyte-binding substance for a non-nucleic acid analyte by adjustingthe reaction conditions of said assay or method to accommodate thespecific analyte and analyte-binding substance. Thus, the methods andassays of the invention permit detection and quantification of anyanalyte for which there is a suitable analyte-binding substance thateither comprises or to which a target sequence tag can be attached. Twomethods for detecting an analyte using an analyte-binding substancecomprising an antibody having a target sequence tag are illustrated inFIGS. 27 and 28.

[0562] S. Use of Transcription Substrates and RNA Polymerases of theInvention as Signaling Systems

[0563] The invention also comprises methods, compositions and kits forusing ssDNA transcription substrates and RNA polymerases that cantranscribe said ssDNA transcription substrates as a signaling system foran analyte of any type, including analytes such as, but not limited to,antigens, antibodies or other substances, in addition to an analyte thatis a target nucleic acid.

[0564] Thus, the invention comprises a method for detecting an analytein or from a sample, said method comprising:

[0565] 1. providing a transcription signaling system, said transcriptionsignaling system comprising a ssDNA comprising: (a) a 5′-portioncomprising a sequence for a promoter for an RNA polymerase that cansynthesize RNA using a ssDNA transcription substrate chosen from amongN4 vRNAP, N4 mini-vRNAP, and N4 mini-vRNAP Y678F enzymes or anotherenzyme that can use a promoter having similar properties; and (b) asignal sequence, wherein said signal sequence, when transcribed by saidRNA polymerase, is detectable in some manner;

[0566] 2. joining said transcription signaling system, either covalentlyor non-covalently, to an analyte-binding substance, wherein said joiningto said substance is not affected by the conditions of the assay andwherein said joining to said substance does not affect the ability ofsaid transcription signaling system to be transcribed using said RNApolymerase under transcription conditions;

[0567] 3. contacting said analyte-binding substance to which saidtranscription signaling system is joined with a sample under bindingconditions, wherein said analyte, if present in said sample, binds tosaid analyte-binding substance so as to form a specific binding pair;

[0568] 4. removing said specific binding pair from said sample so as toseparate it from other components in said sample;

[0569] 5. incubating said specific-binding pair under transcriptionconditions with an RNA polymerase, wherein said RNA polymerase cansynthesize RNA that is complementary to said signal sequence in saidssDNA transcription signaling system under said transcriptionconditions;

[0570] 6. obtaining an RNA synthesis product that is complementary tosaid signal sequence in said ssDNA transcription signaling system; and

[0571] 7. detecting said RNA synthesis product or a substance thatresults from said RNA synthesis product.

[0572] An analyte or an analyte-binding substance of this aspect of theinvention can be any of those described in U.S. Pat. No. 6,562,575,which is incorporated herein by reference. By way of example, but not oflimitation, an analyte-binding substance can be an antibody and theanalyte an antigen, or an analyte-binding substance can be a nucleicacid and the analyte can be another complementary nucleic acid. A largenumber of other substances exist for which a specific-binding pair canbe found. Also the signal sequence can vary greatly. By way of example,but not of limitation, a signal sequence can comprise a substrate forQ-beta replicase, which is detectable in the presence of said replicaseunder replication conditions. It can also comprise a sequence thatencodes a protein, such as green fluorescent protein, that is detectablefollowing translation of the signal sequence. Without limitation, it canalso comprise a sequence that is detectable by a probe, such as, but notlimited to a molecular beacon, as described by Tyagi et al. (U.S. Pat.Nos. 5,925,517 and 6,103,476 of Tyagi et al. and 6,461,817 of Alland etal., all of which are incorporated herein by reference). The presentinvention with regard to signaling systems also comprises uses such asthose for methods described by Zhang et al. (Proc. Natl. Acad. Sci. USA,98: 5497-5502, 2001, incorporated herein by reference) or by Hudson etal. in U.S. Pat. No. 6,100,024, incorporated herein by reference.

[0573] T. Modes of Performance of Methods and Assays of the Inventionfor Detecting a Target Sequence

[0574] Depending on the application and its requirements andconstraints, the methods of the invention can be performed in a stepwisefashion, with one set of reactions being performed, followed bypurification of a reaction product or removal of reagents orinactivation of enzymes or addition of reagents before proceeding to thenext set of reactions. Alternatively, the methods of the invention canbe performed in a preferred embodiment as a continuous set of multiplereactions in a single reaction mixture. The invention also comprisesmethods or assays in which multiple target probes or target probe setsare used in a single reaction mixture in order to detect and/or quantifymultiple target sequences in one or multiple target nucleic acids. Thus,the compositions, kits, methods and assays of the invention can be usedin a multiplex format.

[0575] The invention also comprises parts or subsets of the methods andcompositions of the invention. Thus, the invention comprises all of theindividual steps of the methods of the invention that are enabledthereby, in addition to the overall methods.

[0576] U. Kits and Compositions of the Invention for Detecting a TargetSequence in a Target Nucleic Acid Analyte or a Target Sequence TagComprising or Attached to an Analyte-Binding Substance

[0577] The present invention also comprises kits and compositions forcarrying out the methods of the invention. A kit of the inventioncomprises one or, preferably, multiple components or compositions forcarrying out the various processes of a method. Different embodiments ofkits and compositions of the present invention can comprise one or moreof the following:

[0578] 1. A bipartite target probe for an assay or method for detectinga particular target sequence, and, optionally if thetarget-complementary sequences of said bipartite target probe are notcontiguous when annealed to a target sequence, a monopartite targetprobe, all of which target probes preferably have a 5′-phosphate group.

[0579] 2. A set of monopartite target probes for an assay or method fordetecting a particular target sequence, wherein said set of monopartitetarget probes comprises a promoter target probe, preferably having a5′-phosphate group, and either a signal target probe or a simple targetprobe and one or more additional simple target probes, which if present,preferably each have a 5′-phosphate group.

[0580] 3. A ligase, wherein said ligase has little or no activity inligating blunt ends and is substantially more active in ligating theends of target probe if said ends are adjacent when annealed to twocontiguous regions of a complementary sequence than if said ends are notannealed to said complementary sequence. In preferred embodiments of theabove methods or assays for detecting a target sequence, said ligasecomprises a ligase chosen from among Ampligase® thermostable DNA Ligase,Tth DNA Ligase, Tfl DNA Ligase, Tsc DNA Ligase, or Pfu DNA Ligase.

[0581] 4. An RNA polymerase preparation, wherein said RNA polymeraserecognizes a single-stranded transcription promoter and initiatestranscription therefrom using as a template single-stranded DNA to whichsaid promoter is functionally attached. In some embodiments, said RNApolymerase preparation comprises a single strand binding protein, whichis preferably EcoSSB Protein. In preferred embodiments of the abovemethods or assays for detecting a target sequence, said RNA polymeraseis N4 mini-vRNAP. In some embodiments that incorporate non-canonical2′-modified nucleotides, the N4 mini-vRNAP Y678F mutant enzyme ispreferred.

[0582] 5. A reverse transcriptase, for embodiments that use a reversetranscription process in order to obtain additional amplification of atarget sequence and/or a signal sequence. In preferred embodiments, saidreverse transcriptase has RNase H activity and is chosen from amongMMLV, AMV or another retroviral reverse transcriptase, or a reversetranscriptase encoded by a thermostable phage. In other embodiments,said reverse transcriptase comprises a DNA polymerase chosen from amongIsoTherm™, Bst large fragment, BcaBEST™, and Tth DNA polymerase.Embodiments that use a reverse transcription process also use one ormore primers, which can also comprise a composition or kit of theinvention.

[0583] 6. A DNA polymerase, for embodiments that use DNA polymeraseextension to fill a gap between target probes. Any DNA polymerase thatdoes not strand displace a downstream target probe can be used in acomposition or kit of the invention.

[0584] 7. An analyte-binding substance that either comprises or has anattached target sequence tag for embodiments of the invention fordetecting and/or quantifying an analyte in a sample.

[0585] 8. Compositions of the invention and kits comprising the same fordetecting an RNA transcript that is complementary to a target sequenceand/or a signal sequence. By way of example, but not of limitation, acomposition or kit can comprise a detection oligo, such as a molecularbeacon. Alternatively, a composition or kit can comprise an enzyme, suchas Q-beta replicase, if the signal sequence encodes a Q-beta replicasesubstrate. The invention comprises kits comprising any suitabledetection composition.

[0586] 9. Controls, including quantification standards. Controls areused in assays and methods of the invention in order to verify that theassay or method produces the required specificity and sensitivity, or,in other words, to determine the frequency and conditions that lead to“false positive” and/or “false negative” results. Thus, controlscomprise important compositions and kits for assays and methods of theinvention. By way of example, but not of limitation, a positive controlmight be a sample containing a known quantity of a target sequence. Anegative control would lack the target sequence. For an assay or methodto detect a target nucleotide that is a single nucleotide polymorphismor SNP, positive controls might comprise sample that contain either themutant or the predominant allele or other known alleles for thatnucleotide position in the target sequence. In general, quantificationof a target analyte in a sample using an assay or method of theinvention, including an analyte comprising a target sequence, isachieved by using controls containing different known quantities of saidanalyte as a standard. Provided that the control sample is as close inperformance as possible to a “real world” sample using the methods orassays of the invention, the amount of the analyte in the sample can bestandardized against the results obtained using quantification controls.A composition or kit can also, for example, comprise a controlcomprising an antigen for an assay or method that uses ananalyte-binding substance comprising an antibody with a bound targetsequence tag or a molecule selected using SELEX. Most of the embodimentsfor detecting a target sequence are linear and the side-by-side resultsobtained compared to quantification standards will be proportional tothe amount of said analyte in a sample. However, special care will needto be taken in trying to quantify the amount of analyte in a sample whenan embodiment of an assay or method that comprises secondary oradditional amplification processes, such as, the embodiments illustratedin FIG. 26.

[0587] In general, a kit of the invention will also comprise adescription of the components of said kit and instructions for their usein a particular process or method or methods of the invention. Ingeneral, a kit of the present invention will also comprise othercomponents, such as, but not limited to, buffers, ribonucleotides and/ordeoxynucleotides, including modified nucleotides in some embodiments,DNA polymerization or reverse transcriptase enhancers, such as, but notlimited to betaine (trimethylglycine), and salts of monvalent ordivalent cations, such as but not limited to potassium acetate orchloride and/or magnesium chloride, enzyme substrates and/or cofactors,such as, but not limited to, ATP or NAD, and the like which are neededfor optimal conditions of one or more reactions or processes of a methodor a combination of methods for a particular application. A kit of theinvention can comprise a a set of individual reagents for a particularprocess or a series of sets of individual reagents for multipleprocesses of a method that are performed in a stepwise or serial manner,or a kit can comprise a multiple reagents combined into a singlereaction mixture or a small number of mixtures of multiple reagents,each of which perform multiple reactions and/or processes in a singletube. In general, the various components of a kit for performing aparticular process of a method of the invention or a complete method ofthe invention will be optimized so that they have appropriate amounts ofreagents and conditions to work together in the process and/or method.

[0588] V. Additional Embodiments of the Invention

[0589] Some of the additional embodiments of the invention describedbelow use a ligation splint or a ligation splint oligo. A “ligationsplint” or a “ligation splint oligo” is an oligo that is used to providean annealing site or a “ligation template” for joining two ends of onenucleic acid (i.e., “intramolecular joining”) or two ends of two nucleicacids (i.e., “intermolecular joining”) using a ligase or another enzymewith ligase activity. The ligation splint holds the ends adjacent toeach other and “creates a ligation junction” between the5′-phosphorylated and a 3′-hydroxylated ends that are to be ligated.

[0590] 1. Obtaining a Circular Transcription Subtrate by Circularizing aLigation Product Obtained Using Monopartite Target Probes

[0591] In some embodiments of the present invention, a circulartranscription substrate is obtained using monopartite target probesrather than a bipartite target probe. In these embodiments, themonopartite target probes anneal to the target sequence and are ligatedin the presence of a target sequence to form a linear ligation productas described previously. However, in these embodiments, the linearligation product is denatured from the target sequence and subsequentlycircularized by ligation of its 3′-end to its 5′-end. The 5′-end of thelinear ligation product has a 5′-phosphate group or is phosphorylatedusing a kinase, such as but not limited to T4 polynucleotide kinase, inthe presence of ATP. This 5′-phosphorylated linear ligation product isthen complexed with a ligation splint oligo that has ends that arecomplementary to the 3′- end and the 5′-end of the linear ligationproduct and the ends are ligated under ligation conditions with a ligasethat has little or no activity in ligating blunt ends and that issubstantially more active in ligating ends that are adjacent whenannealed to a contiguous complementary sequence than if the ends are notannealed to the complementary sequence, such as but not limited toAmpligase® DNA Ligase (EPICENTRE Technologies, Madison, Wis.). The useof a ligation splint and a ligase, such as Ampligase® DNA Ligase, thatis not active in ligating blunt ends or non-homologous ligationminimizes “background,” such as background rolling circle transcriptionthat could result from a circular molecule obtained by intramolecularligation of a promoter target probe if a non-homologous ligase wereused. Preferably, the same ligase is used both for ligation of thetarget probes annealed to the target sequence and for subsequentligation of the 3′-end to the 5′-end of the ligation product using aligation splint. After annealing an anti-sense promoter oligo to thecircularized ligation product, a circular transcription substrate of theinvention is obtained.

[0592] One reason to circularize a linear ligation product obtained frommonopartite target probes is because rolling circle transcription isoften more efficient and generates more transcription product thantranscription of linear transcription substrates. Since initiation oftranscription (rather than elongation) is usually a rate-limiting stepfor transcription, the efficiency of transcription of circular versuslinear transcription substrates is particularly increased for smalltranscription substrates. Still further, transcription is also greatlyenhanced for circular transcription substrates in embodiments that usean N4 mini-vRNAP because the transcription product is not efficientlydisplaced from linear transcription substrates (Davidova, E K andRothman-Denes, L B, Proc. Natl. Acad. Sci. USA 100:9250-9255, 2003),whereas the transcription product of rolling circle transcription ofsmall circular transcription substrates by an N4 min-vRNAP is displacedand transcription is therefore much more efficient and productive.

[0593] 2. Use of Probes Lacking a Target-Complementary Sequence

[0594] The invention also comprises embodiments in which a circulartranscription substrate comprising a target-complementary sequence isgenerated even if there is no target-complementary sequence at eitherthe 3′-end or the 5′-end or at both ends of an “open circle probe” (theword “target” is removed from the name of the probe here because thereare no target-complementary sequences). In these embodiments, simpletarget probes that can anneal to the target sequence can be used andthen, following ligation of these simple target probes on the targetsequence, the resulting target-complementary sequence generated byligation of the simple target probes can be joined directly to an opencircle probe by using two ligation splints, each of which has a portioncomplementary to a respective end of the open circle probe and to anappropriate end of the target-complementary sequence. One ligationsplint oligo is used to join a sense promoter of an open circle probe tothe 3′-end of a polynucleotide ligation product that was previouslyobtained by ligation of two or more simple target probes that wereannealed to a target sequence. This first ligation splint oligo has a3′-sequence that is complementary to the 3′-end of the polynucleotideand a second adjacent 5′-sequence that is complementary to the 5′-end ofa the 5′-phosphorylated sense promoter sequence of the open circleprobe. The second ligation splint oligo has a 5′-sequence that iscomplementary to the 5′-end of the polynucleotide and a second adjacent3′-sequence that is complementary to the 3′-end of the open circleprobe. Ligases that can be used to ligate suitable ends that areannealed to a ligation splint comprising DNA include, but are notlimited to, Ampligase® DNA Ligase (EPICENTRE Technologies, Madison,Wis.), Tth DNA ligase, Tfl DNA ligase, Tsc DNA ligase (Prokaria, Ltd.,Reykjavik, Iceland), or T4 DNA ligase. These ligases can be used forboth intermolecular and intramolecular ligations when a ligation splintcomprising DNA is used to bring the respective ends adjacent to eachother. If a ligation splint comprising RNA is used, T4 DNA ligase can beused to join the ends that are annealed to the ligation splint. Theseembodiments of the invention remove all background transcription thatcould result from run-off transcription of the smalltarget-complementary sequence at the 5′-end of a bipartite target probe.

[0595] The invention also comprises similar embodiments for generatinglinear transcripton substrates of the invention, meaning, for example,that simple target probes can be used without using a promoter targetprobe, and/or a signal target probe, and then, the resultingtarget-complementary sequence can be joined to a suitable sense promoterand a signal sequence, if used, by means of ligation splints and aligase under ligation conditions.

[0596] Still further, in other embodiments, simple target probes thatanneal adjacently on a target sequence are ligated, then denatured fromthe target sequence, then ligated to an oligo comprising a sensepromoter sequence using a ligation splint that is complementary to themost 3′-end of the target-complementary target probes and to the 5′-endof a sense promoter sequence, and finally circularized by non-homologousintramolecular ligation of a 5′-phosphorylated end with a 3′-hydroxylend to obtain a circular transcription substrate. Circularization of alinear single-stranded DNA without a ligation splint can be carried outusing ThermoPhage™ RNA Ligase II (Prokaria, Ltd., Reykjavik, Iceland). Areason to circularize a linear ligation product is to obtain a circulartranscription substrate for more efficient transcription by a rollingcircle transcription mechanism, rather than by linear transcription.This embodiment is used only if steps are taken to assure that onlyligation products derived from the target-complementary sequences thatwere ligated in the presence of the target sequence are circularized bythe ligase that catalyzes non-homologous ligation, or that the othernon-target-dependent transcripton products will not be detected in theassay or method.

[0597] 3. Use of Circular Transcription Substrates to SynthesizeDouble-Stranded RNA by Rolling Circle Transcription that can be Used forRNA Interference

[0598] If a target sequence in a target nucleic acid is present in asample, the methods of the invention that use a bipartite target probe,as disclosed herein, can be used to generate a circular transcriptionsubstrate of the present invention. This circular transcriptionsubstrate can be used as a substrate for rolling circle transcription byan RNA polymerase that binds to a single-stranded promoter andsynthesizes RNA therefrom in order to synthesize double-stranded RNA(dsRNA) that can be used to silence a gene by RNA interference. That isthe dsRNA is used as RNAi. By way of example, but not of limitation,dsRNA for use as RNAi can be synthesized using the embodiment of theinvention illustrated in FIG. 26. If the target sequence comprises atarget nucleic acid that is encoded by a pathogen or by an oncogene, forexample, the dsRNA can be a therapeutic composition.

[0599] In another embodiment, a new circular transcription substrate isprepared for synthesis of dsRNA for use as RNAi, wherein each oligomerof the RNA multimer transcription product obtained using said circulartranscription substrate for rolling circle transcription comprises aself-complementary double-stranded hairpin structure with anon-complementary loop between the self-complementary regions, such thateach oligomer corresponds to the desired RNAi and the loop structure.Preferably, said circular transcription substrate is designed so thatsaid RNA oligomers can be cleaved from the RNA multimer obtained fromrolling circle transcription.

[0600] Preferred RNA polymerases for rolling circle transcriptioncomprise the N4 mini-vRNAP and the N4 mini-vRNAP-Y678F enzymes and thepromoter is an N4. Most preferred embodiments of this aspect of theinvention use an enzyme chosen from among N4 mini-vRNAP Y678F mutantenzyme, These most preferred embodiments use an N4 promoter in circulartranscription substrates for N4 mini-vRNAP Y678F mutant enzyme. Mostpreferred embodiments of of this aspect of the invention synthesize RNAcontaining 2′-fluoro-pyrimidine nucleotides by using 2′-fluoro-dCTP and2′-fluoro-dUTP, in addition to ATP and GTP in the rolling circletranscription reaction. Modified RNA molecules that contain 2′-F-dCMPand 2′-F-dUMP are resistant to RNase A-type ribonucleases (Sousa et al.,U.S. Pat. No. 5,849,546), included herein by reference. Capodici et al,(J. Immunology, 169: 5196-5201, 2002) showed that 2′-fluoro-containingdsRNA molecules made using the DuraScribe™ Transcription Kit (EpicentreTechnologies, Madison, Wis., USA) did not require transfection reagentsfor delivery into cells, even in the presence of serum. Kakiuchi et al.(J. Biol. Chem., 257: 1924-1928, 1982) showed that use of[(2′-F-dI)_(n): (2′-F-dC)_(n) duplexes were 40-100 times less antigenicthan [(rI)_(n): (rC)_(n)] duplexes, and did not induce an interferonresponse like [(rI)_(n): (rC)_(n)] duplexes.

EXAMPLES

[0601] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1

[0602] Identification of a Transcriptionally Active Domain of N4 VirionRNA Polymerase

[0603] To determine the minimal domain possessing RNA polymeraseactivity, controlled proteolysis was performed followed by catalytic(transcriptional) autolabeling (Hartmann, et al., 1988). Upon incubationof RNA polymerase with a benzaldehyde derivative of the initiatingnucleotide, the benzaldehyde group forms a Schiff-base with the ε-aminogroup of lysines located within 12 Å of the nucleotide-binding site. Thecrosslinking step was performed in the presence of DNA template becauseit stimulates binding of the initiating nucleotide. The unstableSchiff-base is converted to a stable secondary amine by reduction undermild conditions with sodium borohydride, with concomitant reduction ofany non-reacted benzaldehyde derivative. Addition of the nexttemplate-directed α-³²P labeled NTP leads to phosphodiester bondformation and catalytic autolabeling of the transcriptionally activepolypeptide. Controlled trypsin proteolysis of vRNAP was performed,followed by catalytic autolabeling and analysis on SDS-PAGE (FIG. 3A).Initially, three proteolytic fragments are generated, of which thesmaller two are catalytically active. Upon further incubation withtrypsin, a single stable, transcriptionally active product approximately1,100 amino acids in length remains. N-terminal sequencing of the threeinitial proteolytic fragments (FIG. 3B) indicated that the stable activepolypeptide (mini-vRNAP) corresponds to the middle ⅓ of vRNAP, theregion containing the three motifs described above (FIG. 2A, SEQ IDNOS:3-4).

Example 2

[0604] Cloning and Purification of N4 mini-vRNAP

[0605] The full-size vRNAP and the mini-vRNAP (SEQ ID NOS:6 and 15) ORFswere cloned under pBAD control with an N-terminal hexahistidine tag(FIG. 4). The mini-vRNAP domain was cloned into the pBAD B expressionplasmid, which was purchased from Invitrogen. Five restriction enzymesites within pBAD B have been altered; the SnaI site was converted to aHpaI site, and the PflMI and EcoRV sites were destroyed, all bysite-directed mutagenesis. The BstBI and HindIII sites were destroyed byenzyme digestion followed by Klenow treatment and re-ligation. FIG. 5(left) shows the relative amounts of full-length and mini-vRNAP proteinspurified on TALON columns from the same volume of E. coli BL21 inducedcells. Cloned mini-vRNAP is expressed at 100-fold higher levels thancloned full size vRNAP. Further concentration on a MonoQ column revealsthat, in contrast to full size vRNAP, mini-vRNAP is stable afterinduction (FIG. 5, right). At least 10 mg of mini-vRNAP at a 20 mg/mlconcentration are obtained from 1 L of induced cells in just twopurification steps: TALON and MonoQ minicolumns. A non-histagged versionof mini-vRNAP has also been cloned (SEQ ID NO:4). In this case, theenzyme is purified from a crude extract of induced cells in two steps: apromoter DNA-affinity column and MonoQ.

[0606] Mini-vRNAP possesses a high binding affinity (Kd=1 nM) for N4promoter-containing DNA oligonucleotides. This property was used forpurification of non-his tagged mini-vRNAP (SEQ ID NO:4) on aDNA-affinity column. The column was prepared by adsorbing a 5′biotinylated N4 promoter-containing DNA oligonucleotide onto the matrixof a 1 ml HiTrap Streptavidin column (Pharmacia/AmershamCat.#17-5112-01) according to the manufacturer's instructions. Adebris-free sonicate of bacterial cells expressing mini-vRNAP was passedthrough the column. To bind mini vRNAP to the DNA-affinity column, thepH in the extract and binding/washing buffer should be between 5 to 9,and the NaCl concentration should be between 50 mM and 2M. Nucleases inthe extract are inhibited by addition of 2 mM EDTA. After washing thecolumn, mini-vRNAP was eluted with warm (25° C.) water; the elutiontemperature was raised from 4° C. to 25° C. to increase mini-vRNAPrecovery. For complete elution, the temperature can be raised up to 43°C. without significant change in the quality of the preparation. Elutionunder these conditions occurs due to the removal of metal ions andconsequent melting of the promoter hairpin and dissociation ofmini-vRNAP. Different DNA oligonucleotides containing variants of the P2promoter (SEQ ID NOS:16-19), were used in DNA-affinity columns andtested in mini-vRNAP affinity purification. The best yield was achievedusing the DNA oligonucleotide of SEQ ID NO:16. However, the DNAoligonucleotides of SEQ ID NOS:19-20 require a lower temperature thanthe DNA oligonucleotide of SEQ ID NO:16 for complete elution of theprotein, in agreement with the lower thermal stability of the respectivepromoter hairpins.

[0607] Up to 1 mg of mini-vRNAP of 90% purity is obtained from a crudeextract of 100 ml E. coli culture expressing mini-vRNAP in a singlepurification step using a 1 ml DNA-affinity column. The binding capacityof the DNA-affinity column was not detectably decreased by multiple use.

Example 3

[0608] Effect of EcoSSB on Transcription of Single-Stranded Templates

[0609] Inventors have previously shown that EcoSSB is required for N4vRNAP transcription in vivo (Glucksmann, et al., Cell 70:491-500, 1992).EcoSSB is unique in that, unlike other SSBs whose effect on vRNAPtranscription was tested, it does not melt the promoter hairpinstructure (Glucksmann-Kuis, et al., Cell 84:147-154, 1996). Recently,inventors have reinvestigated the effect of EcoSSB on vRNAPtranscription of single-stranded templates. FIG. 6 shows transcriptionin the absence and presence of Eco SSB at three different ssDNA templateconcentrations. The extent of EcoSSB activation istemplate-concentration dependent, with highest activation at low DNAtemplate concentration. These results suggest that EcoSSB overcomestemplate limitation on ssDNA templates.

[0610] To further explore this hypothesis, the effect of addition oftemplate or EcoSSB to transcription reactions after 20 min incubation inthe absence of EcoSSB was tested. The transcription reaction mixtures(5-50 μl) contained 20 mM Tris-HCl (pH 7.9 at 25° C.), 10 mM MgCl₂, 50mM NaCl, 1 mM dithiothreitol, 0.01-1 μM mini-vRNAP, 1-100 nM ssDNAtemplate (30-100 nt long, synthesized by Integrated DNA Technologies), 1mM each of 3 non-labeled NTPs, 0.1 mM α-³²P NTP (1-2 Ci/mmol, NEN), and1-10 μM E. coli SSB. Incubation was for 1 to 80 min at 37° C. at theindicated temperature. In the presence of EcoSSB, RNA synthesisincreased linearly throughout the period of incubation (FIG. 7C). In theabsence of EcoSSB, no increase in transcription was observed beyond 10min of incubation (FIG. 7A). Addition of template at 20 min to thereaction carried out in the absence of EcoSSB led to a dramatic increasein RNA synthesis (FIG. 7B). Addition of EcoSSB at 20 min led to a slowrate of transcriptional recovery (FIG. 7D). These results suggest thatEcoSSB converts the template from a transcriptionally inactive RNA:DNAhybrid to transcriptionally active single-stranded DNA.

[0611] To test this hypothesis, the physical states of the DNA templateand the RNA product were analyzed by native gel electrophoresis in theabsence and in the presence of EcoSSB. In order to have effectivetranscription in the absence of EcoSSB, transcription was performed atan intermediate (5 nM) DNA concentration, at which only a 2-fold effectof EcoSSB is observed.

[0612] The results of this experiment are shown in FIG. 8. Either³²P-labeled template (right panel) or labeled NTPs (left panel) wereused to analyze the state of the template (right panel) or RNA product(left panel) in the absence or presence of EcoSSB. After transcription,the mixtures were split further into 3 samples: a control sample with noadditions, a sample to which RNase H was added to specifically degradeRNA in RNA:DNA hybrids, and a third sample to which Nuclease S1 wasadded to degrade single-stranded nucleic acids. In the absence ofEcoSSB, both the DNA template and the RNA product are in RNA:DNAhybrids, since the RNA product is RNase H sensitive while theDNA-containing bands show altered mobility after RNase H treatment. Inthe presence of EcoSSB, a significant portion of the RNA product isRNase H resistant and therefore free, although an RNase sensitive bandis present that corresponds to an intermediate RNA:DNA:SSB complex.Under these conditions, the DNA is in an SSB:DNA complex. These resultsindicate that EcoSSB stimulates transcription through templaterecycling.

[0613] To define regions of EcoSSB essential for vRNAP transcriptionactivation on single-stranded templates, the inventors have tested theeffect of human mitochondrial SSB (HmtSSB), which shows extensivesequence and structural homology to EcoSSB. The N-terminus of EcoSSBcontains DNA binding and tetramerization determinants while theC-terminus is involved in interaction with other replication proteins.Hmt SSB has no effect on vRNAP transcription although it does not meltthe promoter hairpin. Interestingly, preliminary results using mutantEcoSSBs and EcoSSB-Hmt SSB chimeras suggest that the C-terminal regionof EcoSSB is essential for vRNAP transcriptional activation.

Example 4

[0614] Characterization of mini-vRNAP Transcription Properties

[0615] The initiation properties of the full length RNA polymerase andmini-vRNAP were compared at similar molar concentrations (FIG. 9A) usingthe catalytic autolabeling assay and two reaction conditions: 1-using atemplate containing +1C, the benzaldehyde derivative of GTP andα³²P-ATP, or 2- a template containing +1T, the benzaldehyde derivativeof ATP and α³²P-GTP. Comparison of the results in FIGS. 9B and 9Cdemonstrates that mini-vRNAP exhibits initiation properties similar tofull-length vRNAP. In addition, both enzymes discriminate against DATPincorporation to the same extent. Mini-vRNAP does not synthesizeabortive products when the first four nucleotides of the transcript arecomprised of 50% or more G or C nucleotides.

[0616] The elongation and termination properties of both enzymes arecompared in FIG. 10. Similar run-off and terminated transcripts aresynthesized. Moreover, EcoSSB activates transcription by both enzymes tothe same levels. This result indicates that, if there are any sites ofspecific contact between vRNAP and EcoSSB, they reside in the mini-vRNAPdomain.

[0617] The sequence of the terminator signals for vRNAP present in theN4 genome include SEQ ID NOS:21-26. The signals of SEQ ID NO:21 and 22have been tested in vitro on single-stranded templates.

[0618] The rate of mini-vRNAP transcription has been compared to therate of T7 RNA polymerase under the same conditions using the same DNAtemplate. The template used was linearized pET11 containing the originalT7 promoter and the N4 vRNAP P2 promoter that was introduced throughcloning. The DNA template was denatured before performing transcriptionusing N4 mini-vRNAP. The concentrations of T7 RNAP (Promega, Cat.#P2075)and mini-vRNAP were compared using SDS-PAGE. Transcription reactionscontained 50 nM of polymerase, 100 nM of DNA template, 5×transcriptionbuffer provided with the T7 RNAP, and 1 mM of each ATP, GTP and CTP and0.1 mM of [³²p]-UTP (1 Ci/mmol). Each reaction mixture was split in two,and E. coli SSB was added to one half. The mixtures were incubated at37° C. and aliquots were taken at different time points. Transcriptionproducts were electrophoresed on a 6% sequencing gel and the amount ofradioactively-labeled RNA was quantitated by phosphoimaging. The resultsshowed that: (a) transcription of T7 RNAP was not affected by thepresence of E. coli SSB and (b) N4 mini-vRNAP synthesized 1.5 to 5 foldmore RNA in the presence of EcoSSB than T7 RNAP at different time pointsof incubation.

[0619] The optimal temperature for mini-vRNAP transcription is 37° C. Itexhibits 70% activity at 30° C., 65% at 45° C., and only 20% at 50° C.

[0620] The average error frequency was estimated by determining themisincorporation frequency of each of four [32P]-α NTPs into RNAproducts using template ssDNAs missing the corresponding templatenucleotide in the transcribed region. The following values wereobtained: ⅕×10⁴ for misincorporation of G and U using “no C” (SEQ IDNO:10) and “no A” (SEQ ID NO:11) ssDNA templates, respectively; ¼10⁴ formisincorporation of C using the “no G” (SEQ ID NO:12) template, and½×10⁴ for misincorporation of A using the “no T” (SEQ ID NO:13)template. For comparison, the average error frequency for T7 RNAP is½10⁴ (Huang, et al., 2000). Using the method for detection of mispairformation described by Huang, et al. (2000), no misincorporation bymini-vRNAP was detected.

[0621] The ability of mini-vRNAP to incorporate derivatized nucleotideswas measured. Transcription by mini-vRNAP in the presence of 0.1-1 mMDigoxigenin-11-UTP (cat# 1209256, Roche), Biotin-16-UTP (cat# 1388908,Roche) or underivatized UTP, yielded comparable amounts of product RNAusing “control” ssDNA (SEQ ID NO:9) as a transcription template. Theproduct RNAs synthesized in the presence of derivatized UTP have highermolecular mass than those synthesized in the presence of underivatizedUTP, and the difference corresponds to the mass difference of the UTPsused. Several derivatives (i.e. 2° Fluoro-ribonucleoside triphosphates,dideoxynucleoside triphosphates) are being tested. The fluorescentanalog Fluorescein-12-UTP (Roche catalog #1427857) has been tested usinga template which encodes a 51 nucleotide transcript containing a run of4 Us, and a nucleotide mix containing ATP, CTP, GTP andFluorescein-12-UTP only. Transcription was only 3% of that achieved withUTP, biotin-6-UTP or digoxigenin-11-UTP under the same reactionconditions. However, incorporation of the fluorescent analog at higheryields is expected to occur in the presence of underivatized UTP or ontemplates with other sequence compositions.

Example 5

[0622] Sequence Determinants of mini-vRNAP Promoter Binding

[0623] The three N4 early promoters present in the N4 genome contain apair of Cs separated by 4 nucleotides from the base of the 5 bp promoterstem. In the preferred promoter P2, these 4 bases are As and the Cs arefollowed by a T. Preferably, mini-vRNAP uses a 17 nucleotide promotersequence located immediately upstream of the transcription initiationsite. Promoters for N4 vRNA polymerase are described by Haynes et al.,Cell 41:597-605 (1985) and Dai et al., Genes Devepmnt. 12:2782-2790(1998), herein incorporated by reference. vRNAP-promoter recognition andactivity require specific sequences and a hairpin structure on thetemplate strand. The vRNAP promoters of SEQ ID NOS:27-29 assume ahairpin structure comprised of a 5-7 bp stem and 3 b purine-containingloop. The −11 position corresponds to the center of the loop; +1indicates the transcription start site. Thus, promoter sequences of theinvention include, but are not limited to: SEQ ID NO:27 −11 +1 P13′-CAACGAAGCGTTGAATACCT-5′ SEQ ID NO:28 −11 +1 P23′-TTCTTCGAGGCGAAGAAAACCT-5′, and SEQ ID NO:29 −11 +1 P33′-CGACGAGGCGTCGAAAACCA-5′

[0624] Other possible vRNAP promoters of the current invention include aset of any inverted repeats forming a hairpin with a 2-7 bp long stemand 3-5 b loop having purines in the central and/or next to the centralposition of the loop.

[0625] To study the sequence determinants of promoter binding, 20base-long promoter oligonucleotides, containing the wild-type vRNAPpromoter P2 sequence and substituted at every position with a single5-Iodo-dU, were used. Whenever substitutions were made in the stem, thecorresponding pairing base was changed to A. These oligonucleotides were³²P end-labeled and used to determine the enzyme's affinity for promoterDNAs by a filter binding assay and the ability to crosslink tomini-vRNAP upon UV irradiation at 320 nm. A 20-base oligonucleotide withwild type promoter P2 sequence binds with a 1 nM Kd. Mostoligonucleotides showed close to wild type affinity except for theoligonucleotides substituted at positions −11 (at the center of theloop) and −8, indicating that these positions are essential for promoterrecognition (FIG. 11). Surprisingly, UV crosslinking was most effectiveat position 31 11, in spite of the low binding affinity, indicating aspecific contact at this position to mini-vRNAP. Crosslinking was alsoobserved to positions +1, +2 and +3, indicating non-specific contactswith this region of the template, since 5-Iodo-dU substitutedoligonucleotides at these positions showed wild-type binding affinity.

[0626] The effect of changes in the stem length of the hairpin on theability of mini-vRNAP to bind P2 promoter DNA was analyzed. As shownabove, wild type promoter P2 with a 5 bp stem has a Kd of 1 nM (FIG. 12,top). The stem was shortened by removal of 3′ bases as shown in FIG. 12(left). The stem can be shortened by two base pairs without change inthe binding affinity. If two or one loop-closing base pairs remain, thebinding affinity of templates is still substantial (2-10 nM). Thisresult, although surprising, is not unexpected since it has been shownthat the oligonucleotide 3′d(CGAGGCG)5′ forms an unusually stableminihairpin (Yoshizawa, et al., Biochemistry 36, 4761-4767, 1997). Nobinding is observed if one more nucleotide is removed and the loopcannot form. These results indicate that formation of a loop isessential for vRNAP-promoter recognition.

[0627] The effect of lengthening the stem by addition of 3′ bases isshown in FIG. 12 (right). The stem can be lengthened by two base pairswithout change in the binding affinity. On the other hand, base pairingat −2 reduces binding affinity by two orders of magnitude, with afurther one order of magnitude reduction caused by base pairing at −1and +1. These results indicate that single-strandedness of the templateat positions −2, −1 and +1 is required for efficient template binding.

[0628] All three N4 early promoters present in the N4 genome contain apair of Cs separated by 4 nucleotides from the base of the 5 bp promoterstem. In promoter P2, these 4 bases are As and the Cs are followed by aT. To identify the determinants of the site of transcription initiation,a series of templates were constructed with a single C placed atdifferent distances from position −11 of the hairpin by addition ordeletion of the tract of As present at promoter P2 (FIG. 13). Theaffinity of mini-vRNAP for these promoters was measured by filterbinding and transcription initiation was measured by catalyticautolabeling of mini-vRNAP. All templates showed similar bindingaffinities. However, only the template with a C positioned 12 basesdownstream from the center of the hairpin was able to supporttranscription initiation. This result indicates that mini-vRNAP utilizesthis position as the transcription start site (+1).

Example 6

[0629] Identification of Sequence Motifs Essential for mini-vRNAPActivity

[0630] As shown in FIG. 2A, vRNAP contains the sequence Rx₃Kx₆YG,designated Motif B in the Pol I and Pol α DNA polymerases and theT7-like RNA polymerases. To determine the relevance of this motif tovRNAP activity, two mutants K670A and Y678F (SEQ ID NO:8) (positionnumbers in mini-vRNAP) were constructed by site-specific mutagenesis ofmini-vRNAP. These two positions were chosen because, in T7-like RNApolymerases, the lysine is involved in nucleotide binding and thetyrosine in discrimination against deoxynucleoside triphosphates(Maksimova, et al., Eur. J Biochem. 195:841-847, 1991; Bonner, et al.,EMBO J. 11:3767-3775, 1992; Osumi-Davis, et al., J. Mol Biol. 226:37-45,1992). The His-tagged Y678F mini-vRNAP gene (SEQ ID NO:7) differs fromthat of the mini-vRNAP domain sequence (SEQ ID NO:3) at two positions:nucleotide 2033 (A) was changed to a T, and nucleotide 2034 (T) waschanged to a C.

[0631] These RNA polymerase mutants were cloned under pBAD control,purified and tested for their ability to bind to wild type promoters.Both mutant polymerases bound to promoter DNA with wild type affinitiesand crosslinked to 5-Iodo-dU substituted P2 DNA templates at positions−11 and +3 with wild-type affinities (FIG. 14), indicating that thesemutations do not affect promoter binding.

[0632] The mutant enzymes were tested for their ability to supportrun-off transcription. The wild-type enzyme and Y678F enzyme (SEQ IDNO:8) displayed similar activities at both template excess andtemplate-limiting conditions, while the K670A enzyme exhibited decreasedactivity under both conditions (FIG. 15). Under limiting templateconditions, all three enzymes were activated by Eco SSB (right panel).However, the Y678F enzyme showed reduced discrimination between ribo-and deoxyribonucleoside triphosphates.

[0633] The initiation properties of the three enzymes were comparedusing catalytic autolabeling (FIG. 16). The K670A enzyme displayssignificantly reduced activity with the GTP derivative. The Y678Fenzyme, in contrast to wild-type polymerase, incorporates dATP asefficiently as rATP in a single round of phosphodiester bond formation.

[0634] Therefore, the behavior of the K670A and Y678F mutant enzymesindicates that Motif B is involved in catalysis, with the lysineprobably required for NTP binding and the tyrosine responsible for dNTPdiscrimination. These results suggest that, despite its lack ofextensive sequence similarity, vRNAP is a Class II T7-like RNApolymerase. Results of recent experiments revealed the location of thetwo carboxylates (aspartates) involved in catalysis.

Example 7

[0635] Development of an In Vivo System Using Mini-vRNAP and N4 vRNAPPromoters for in vivo Expression of RNAs and Proteins

[0636] Plasmid templates were constructed with a reporter gene(α-peptide of β-galactosidase) cloned under the control of vRNAPpromoter P2 present in either of two orientations (FIG. 17B). Thereporter construct was generated by cloning a cassette into plasmidpACYC177, which was obtained from New England Biolabs. The cassettecontains an approximately 30 bp long fragment originating from pT7Ac(purchased from United States Biochemical), a N4 promoter, and sequenceencoding the alpha fragment of lacZ (lacZ′). The N4 promoter and lacZ′were generated by oligonucleotide annealing and PCR amplification,respectively. This cassette replaces the pACY177 sequence locatedbetween the cleavage sites for restriction enzymes ApaLI and BamHI.These reporter plasmids and recombinant full-length or mini-vRNAPexpressing plasmids were introduced into E. coli DH5α (ΔM 15), a strainthat encodes the β-galactosidase σ-peptide. Expression of the reportergene α-peptide) in this strain results in the synthesis of activeβ-galactosidase and consequent production of blue colonies on X-galplates. Transcription of α-peptide by full-length and mini-vRNAP wasassayed on inducing-Xgal media and shown in FIG. 17A. Induction offull-length polymerase results in small colonies with no β-galactosidaseactivity. This is not surprising since full-length vRNAP is degraded inthese cells (FIG. 17C). In contrast, induction of mini-vRNAP led todetectable levels of the protein (FIG. 17C) and to β-galactosidaseactivity only from the plasmid containing promoter P2 in the properorientation (FIG. 17A). These results indicate that this system will besuitable for in vivo expression of RNAs and proteins under mini-N4 vRNAPpromoter control.

[0637] All of the methods disclosed and claimed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to the methodsand in the steps or in the sequence of steps of the method describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

Example 8

[0638] Rolling Circle Transcription of Model ssDNA TranscriptionSubstrates

[0639] Each oligonucleotides (50 picomoles), comprising a sense P2promoter sequence (or, in control reactions, an anti-sense sequence tothe P2 promoter or no promoter) at its 5′-end, which was phosphorylated,and up to 52 additional nucleotides corresponding to a model targetsequence (e.g., for the human beta actin gene) in its 3′-portion, wasligated in a reaction mixture containing 0.2 mM ATP, 1 mM DTT, and 50micrograms per ml of BSA for 2 hours at 60° C. using 200 units ofThermoPhage™ RNA Ligase II (Prokaria, Rejkjavik, Iceland, #Rlig122) in1× ThermoPhage RNA Ligase II Buffer comprising 50 mM MOPS, pH 7.5, 5 mMMgCl₂, and 10 mM KCl. Then, linear oligos were removed by digestion withExonuclease I (EPICENTRE Technologies, Madison, Wis.), the Exo I washeat-inactivated, and the circular ssDNA oligos were ethanolprecipitated using standard techniques.

[0640] One picomole of circular ssDNA oligonucleotide, prepared as justdescribed, was then incubated for four hours at 37° C. in a60-microliter reaction mixture comprising one microgram of N4 mini-vRNAP(EPICENTRE Technologies, Madison, Wis.), 1 mM each NTP, 1 mM DTT, and 5micromolar E. coli SSB Protein (EPICENTRE Technologies, Madison, Wis.),in 1× Transcription Buffer comprising 40 mM Tris HCl, pH 7.5, 10 mMNaCl, 6 mM MgCl₂, and 1 mM spermidine. The resulting mini-vRNAPtranscription products were then analyzed by electrophoresis in a 1%agarose gel containing 0.22 M formaldehyde. Transcription products,including products having a length many-fold greater than the startingoligonucleotide, were observed on the the gel using the transcriptionsubstrate having a sense P2 promoter sequence, indicating efficientrolling circle transcription. No transcription products were observed ifthe oligo did not contain a P2 promoter, if an anti-sense sequence tothe P2 promoter was used instead of the sense P2 promoter, or if anunligated linear oligo with a sense P2 promoter was used.

Example 9

[0641] Use of Target-Dependent Transcription Using Bipartite TargetProbes Comprising a P2 Promoter to Detect the Human β-globin GeneSequence in which a Single Nucleotide Mutation Results in Sickle CellAnemia

[0642] Bipartite target probes were designed to anneal to the geneencoding human hemoglobin β chain. The ligation junction of the adjacentprobes when annealed to the denatured globin gene is the site of asingle-base difference responsible for the sickle-cell phenotype (an Ato T transversion leading to GluàVal change in the β-globin). The probecan be circularized by DNA ligase only when annealed to the wild-typeglobin allele, but not when the ligation junction is annealed to atarget nucleotide comprising a single-base mismatch that results in thesickle-cell phenotype.

[0643] Oligonucleotide target probes were obtained from Integrated DNATechnologies, Coralville, IA and were 5′ phosphorylated duringsynthesis. All human β-globin bipartite target probes consisted of twotarget-complementary arms in the 5′ and 3′ terminal regions connected bya spacer of a specific size that contained a P2 promoter sequence, aswell as (optional) binding sites for amplification primers, restrictionsites, signal sequences, etc. The 5′ arm length was from 11 to 18nucleotides and was designed to anneal immediately upstream of thesingle-base mismatch that results in the sickle-cell phenotype. The 3′arm was from 14 to 20 nucleotides long and was complementary to theregion immediately downstream of this mutation. In most probes the3′-terminal base was complementary to the nucleotide that differed inthe wild type and mutant alleles of the β-globin gene. This was done toimprove allele discrimination, since base mismatches at the 3′ terminusare more inhibitory to ligation than those at 5′ terminus (Luo et al.,Nucleic Acids Res., 24:3071-3078, 1996). The length of the spacer regionshould allow circularization of the oligo while its 5′ and 3′ terminalarms are annealed to the target and cannot be shorter then 1.26-timesthe combined length of target-complementary arms.

[0644] The sequence of one of the human β-globin bipartite target probesis given below. The target-complementary sequences are underlined. TheP2 promoter hairpin sequence is in italics.

[0645] 5′Phos-GTCCTCAGTCCCAAAAGAAGCGGAGCTTCT(24)CCGTCTGAAGAGGA3′

[0646] (67 bases, 25 are complementary to the target)

[0647] Bipartite target probes for detection of a beta-globin genesequence were designed with a goal to be optimal for (i) targetrecognition specificity and thermostable ligase activity underhybridization and ligation reaction conditions and (ii) N4mini-vRNAP-catalyzed rolling circle transcription. Thus, the probe wasdesigned so that the P2 promoter hairpin was the only stable secondarystructure at 37° C., and the target-complementary portions of the targetprobes were long enough to hybridize preferentially to the targetsequence at a hybridization temperature that would still providesufficient thermostable ligase activity. At the same time the overalllength of the probe was kept to a minimum (under 100 nucleotides) tounsure efficient rolling circle (RC) transcription.

[0648] The β-globin bipartite target probes were incubated withsubcloned plasmid DNA containing the wild-type β-globin gene sequence,digested with Apa LI restriction endonuclease. Target probes wereannealed and ligated to the target sequence as follows: 2.5 microgramsof plasmid DNA comprising the target sequence were denatured andhybridized to 2 to 50 picomoles of each target probe in 20 mM Tris-HCl(pH 8.3 at 25° C.), 25 mM KCl, 10 mM MgCl2, 0.5 mM NAD, 0.01% TritonX-100 at 94° C. for 1.5 minutes in the total volume of 50 ul. Targetprobes annealed to the target sequence were ligated using 50 Units ofAmpligase® Thermostable Ligase (EPICENTRE Technologies, Madison, Wis.)by thermocycling for 20 to 50 cycles of 94° C. for 30 seconds and 40° C.for 6 minutes. The unligated probe was then removed by digestion with 40units of E. coli Exonuclease I (EPICENTRE) for 30 minutes at 37° C.Ligation reactions were ethanol-precipitated or used directly assubstrates for the N4 mini-vRNAP transcription.

[0649] Transcription reactions were analyzed as follows: 10-25% of theligation reaction was used as template in the transcription reaction.The 20 ul reactions contained 1 mM each NTP, 1 mM DTT, 5 uM EcoSSBProtein (EPICENTRE), 1 U/ul RNasin (Promega, Fitchburg, Wis.), and 8pmol N4 mini-vRNAP (EPICENTRE) in 1×transcription buffer comprising 40mM Tris HCl, pH 7.5, 10 mM NaCl, 6 mM MgCl2, and 1 mM spermidine.Reactions were incubated at 37° C. for 2 to 6 hours. The transcriptionreactions were treated with 2 Units DNAse I for 30 minutes at 37° C.Samples were heat denatured in formamide loading buffer with 0.1% SDSand were analyzed by denaturing 1% agarose gel electrophoresis in 1× TAEbuffer.

[0650] Upon hybridization with the target sequence, a bipartite probeoligo circularizes and serves as efficient template for N4mini-vRNAP-catalyzed rolling circle transcription, yielding highmolecular weight RNA products. The unligated linear probe yields only a11-18-nucleotide transcript (and no signal sequence would be obtained).

[0651] As expected, high molecular weight RNA transcription productswere observed only in reactions in which the wild type β-globin targetprobes were incubated under hybridization and ligation conditions in thepresence of the wild-type β-globin sequence. No high molecular weighttranscription product was observed in the absence of the wild-typeβ-globin sequence, in the absence of ligase, or in the presence of onlyone target probe.

[0652] Various modifications and variations of the described method andsystem of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. It isunderstood, however, that examples and embodiments of the presentinvention set forth above are illustrative and not intended to confinethe invention. The invention embraces all modified forms of the examplesand embodiments as come within the scope of the following claims.

1 29 1 10506 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 1 atgtcagtat ttgatagact ggctgggttc gcagacagcgtaaccaatgc aaagcaagtt 60 gacgtctcta ctgcaaccgc ccagaagaaa gctgaacaaggtgtcactac tcctcttgtt 120 tctcctgatg ctgcttatca aatgcaagct gcccgtactggtaatgttgg ggctaatgca 180 tttgaaccag ggacagtgca atcagatttc atgaatctgaccccaatgca aatcatgaat 240 aagtatgggg ttgagcaagg cttacaactt atcaatgctcgtgctgatgc agggaaccag 300 gtattcaatg attcagttac tacaagaact cctggggaagaactggggga tattgctact 360 ggtgttggcc ttggttttgt taataccctt gggggcattggtgctcttgg ggcaggctta 420 ctcaacgatg atgcaggtgc tgttgttgct caacaattgagtaagtttaa tgatgctgtt 480 catgctaccc aaagccaggc attacaagat aaacgtaagctctttgctgc tcgtaactta 540 atgaatgaag tagagagtga acgtcagtat caaacagataagaaagaagg cactaatgac 600 atagtagctt ccttatctaa atttggacgt gattttgtaggttcaattga gaatgctgct 660 caaactgact ctattatttc tgatgggtta gcagaaggggtaggttctct attaggtgct 720 ggtcctgtat taaggggtgc atctttactg ggtaaagcagttgttccagc aaatactctt 780 cgtagtgctg cattggctgg tgctattgat gcaggtactggtactcagtc actggctcgt 840 attgcctcta ctgtaggtag agctgcaccg ggtatggttggtgttggtgc aatggaagct 900 ggtggtgcat accaacaaac tgctgatgaa attatgaagatgagtcttaa agacttagag 960 aagtctcctg tttatcagca acatattaaa gatggtatgtcccctgaaca ggctcgtcgt 1020 cagactgcat ctgaaactgg tcttactgct gctgctattcaattacctat tgctgctgca 1080 accggtcctc tggtatcccg ttttgagatg gctcctttccgtgctggctc tttaggtgct 1140 gtaggtatga accttgcccg tgaaacagtg gaagaaggtgttcagggtgc tacaggccaa 1200 ctggctcaga atattgcaca gcaacaaaac attgataagaaccaagacct gcttaaaggt 1260 gtcggtacac aggctggttt aggtgctctt tatggctttggttctgctgg tgttgtacag 1320 gctccggctg gtgctgctcg tttagcaggt gctgcaactgctcctgtatt gcgtaccaca 1380 atggctggtg ttaaagctgc tggtagtgta gcaggtaaggttgtttctcc tattaagaat 1440 actttagtag ctcgtggtga acgggttatg aagcagaatgaagaagcatc tcctgttgct 1500 gatgactatg ttgcacaggc agcacaagaa gctatggctcaagcaccaga agcagaagtt 1560 actattcgtg atgctgttga agcaactgat gctactccagaacagaaagt tgcagcacac 1620 cagtatgttt ctgacttaat gaatgctact cgttttaatcctgaaaatta tcaggaagca 1680 ccagagcata ttcgtaatgc tgtagctggt tctactgaccaagtacaggt tattcagaag 1740 ttagcagact tagttaacac attagatgaa tctaatcctcaagcactgat ggaagctgca 1800 tcttatatgt atgatgctgt ttcagagttt gagcagttcattaaccgtga ccctgctgca 1860 ctggatagca ttcctaaaga ttctccggct attgagttactcaaccgtta tacgaatctg 1920 acagctaata ttcagaacac accaaaagta attggtgcactgaatgttat taatcgaatg 1980 attaatgaat ctgctcagaa tggttctttg aatgtgactgaagaatccag tccacaggaa 2040 atgcagaacg tagcattagc tgctgaagta gcccctgaaaagctcaatcc agagtctgta 2100 aatgttgttc ttaaacatgc tgctgatggt cgtattaaactgaataatcg ccagattgct 2160 gccctccaga atgctgctgc aatcctgaag ggggcacgggaatatgatgc agaagctgcc 2220 cgtcttggat tacgtcctca agacattgtg agtaaacagattaaaacgga tgagagcaga 2280 actcaggaag gacaatactc tgcgttgcaa catgcgaataggattcggtc tgcgtataac 2340 tctggtaatt tcgagttggc ctccgcttac ctgaacgactttatgcagtt cgcccagcac 2400 atgcagaata aggttggagc gttgaatgag catcttgttacggggaatgc ggataagaat 2460 aagtctgtcc actaccaagc tcttactgct gacagagaatgggttcgtag ccgtaccgga 2520 ttgggggtca atccctatga cactaagtcg gttaaatttgcccagcaagt tgctcttgaa 2580 gcgaaaacgg tagcggatat tgctaatgcc ctcgcttcggcttacccgga actgaaggtc 2640 agtcatataa aagttactcc attggattca cgtcttaacgctcctgctgc tgaggtggtc 2700 aaggcattcc gtcaaggcaa tcgagacgtt gcttcttctcaaccgaaagc tgactccgtg 2760 aatcaggtta aagaaactcc tgttacaaaa caggaaccagttacatctac tgtacagact 2820 aagactcctg ttagtgaatc tgttaaaaca gaacctactactaaagagtc tagcccacag 2880 gctataaaag aacctgtgaa ccagtctgaa aaacaggatgttaaccttac taatgaggac 2940 aacatcaagc aacctactga atctgttaaa gaaactgaaacttctacaaa agaaagtaca 3000 gttacagaag aattaaaaga aggtattgat gctgtttacccttcattggt aggtactgct 3060 gattctaaag cagagggtat taagaactat ttcaaattgtcctttacctt accagaagaa 3120 cagaaatccc gtactgttgg ttcagaagca cctctaaaagatgtagccca agctctgtct 3180 tctcgtgctc gttatgaact ctttactgag aaagaaactgctaaccctgc ttttaatggg 3240 gaagttatta agcgatacaa agaactcatg gaacatggggaaggtattgc tgatattctt 3300 cgctcccgtc tggctaagtt ccttaacact aaggatgttggtaaacgttt tgctcaaggt 3360 acagaagcca accgttgggt aggtggtaag ttacttaacattgttgagca ggatggggat 3420 acctttaagt acaacgaaca attgctacag actgctgtattagcaggtct tcaatggaga 3480 cttactgcta ccagcaatac tgctatcaaa gatgcaaaagatgttgctgc tattactggt 3540 attgaccaag ctctgctgcc agaaggttta gtagagcaatttgatactgg tatgacactc 3600 actgaagcag ttagttccct ggctcagaaa attgagtcttactggggatt atctcgtaat 3660 ccaaatgctc cattgggcta taccaaaggc atccctacagcaatggctgc tgaaattctg 3720 gctgcatttg tagagtctac tgatgttgta gagaacatcgtggatatgtc agaaattgac 3780 ccagataaca agaagactat tggtctgtac accattactgaactggattc cttcgaccca 3840 attaatagct tccctactgc tattgaagaa gctgttttagtgaatcctac agagaagatg 3900 ttctttggtg atgacattcc tcctgtagct aatactcagcttcgtaaccc tgctgttcgt 3960 aatactccag aacagaaggc tgcattgaaa gcagagcaggctacagagtt ctatgtacac 4020 accccaatgg ttcaattcta tgagacgtta ggtaaagaccgtattctcga actgatgggt 4080 gctggtactc tgaataaaga gttacttaat gataaccatgctaaatctct ggaaggtaag 4140 aaccgttcag tagaggactc ttacaaccaa ctgttctccgtcattgagca ggtaagagca 4200 cagagcgaag acatctctac tgtacctatt cactatgcatacaatatgac ccgtgttggt 4260 cgtatgcaga tgttaggtaa atacaatcct caatcagccaaactggttcg tgaggccatc 4320 ttacctacta aagctacttt ggatttatcg aaccagaacaatgaagactt ctctgcattc 4380 cagttaggtc tggctcaggc attggacatt aaagtccatactatgactcg tgaggttatg 4440 tctgacgagt tgactaaatt actggaaggt aatctgaaaccagccattga tatgatggtt 4500 gagtttaata ccactggttc cttaccagaa aacgcagttgatgttctgaa tacagcatta 4560 ggagatagga agtcattcgt agcattgatg gctcttatggagtattcccg ttacttagta 4620 gcagaggata aatctgcatt tgtaactcca ctgtatgtagaagcagatgg tgttactaat 4680 ggtccaatca atgccatgat gctaatgaca ggcggtctgtttactcctga ctggattcgt 4740 aatattgcca aagggggctt gttcattggt tctccaaataagaccatgaa tgagcatcgc 4800 tctactgctg acaataatga tttatatcaa gcatccactaatgctttgat ggaatcgttg 4860 ggtaagttac gtagtaacta tgcctctaat atgcctattcagtctcagat agacagtctt 4920 ctttctctga tggatttgtt tttaccggat attaatcttggtgagaatgg tgctttagaa 4980 cttaaacgtg gtattgctaa gaacccactg actattaccatctatggttc tggtgctcgt 5040 ggtattgcag gtaagctggt tagttctgtt actgatgccatctatgagcg tatgtctgat 5100 gtactgaaag ctcgtgctaa agacccaaat atctctgctgctatggcaat gtttggtaag 5160 caagctgctt cagaagcaca tgctgaagaa cttcttgcccgtttcctgaa agatatggaa 5220 acactgactt ctactgttcc tgttaaacgt aaaggtgtactggaactaca atccacaggt 5280 acaggagcca aaggaaaaat caatcctaag acctataccattaagggcga gcaactgaag 5340 gcacttcagg aaaatatgct gcacttcttt gtagaaccactacgtaatgg tattactcag 5400 actgtaggtg aaagtctggt gtactctact gaacaattacagaaagctac tcagattcaa 5460 tctgtagtgc tggaagatat gttcaaacag cgagtacaagagaagctggc agagaaggct 5520 aaagacccaa catggaagaa aggtgatttc cttactcagaaagaactgaa tgatattcag 5580 gcttctctga ataacttagc ccctatgatt gagactggttctcagacttt ctacattgct 5640 ggttcagaaa atgcagaagt agcaaatcag gtattagctactaaccttga tgaccgtatg 5700 cgtgtaccaa tgagtatcta tgctccagca caggccggtgtagcaggtat tccatttatg 5760 actattggta ctggtgatgg catgatgatg caaactctttccactatgaa aggtgcacca 5820 aagaataccc tcaaaatctt tgatggtatg aacattggtttgaatgacat cactgatgcc 5880 agtcgtaaag ctaatgaagc tgtttacact tcttggcagggtaaccctat taagaatgtt 5940 tatgaatcat atgctaagtt catgaagaat gtagatttcagcaagctgtc ccctgaagca 6000 ttggaagcaa ttggtaaatc tgctctggaa tatgaccaacgtgagaatgc tactgtagat 6060 gatattgcta acgctgcatc tctgattgaa cgtaacttacgtaatattgc actgggtgta 6120 gatattcgtc ataaggtgct ggataaggta aatctgtccattgaccagat ggctgctgta 6180 ggtgctcctt atcagaacaa cggtaagatt gacctcagcaatatgacccc tgaacaacag 6240 gctgatgaac tgaataaact tttccgtgaa gagttagaagcccgtaaaca aaaagtcgct 6300 aaggctaggg ctgaagtcaa agaagaaact gtttctgaaaaagaaccagt gaatccagac 6360 tttggtatgg taggccgtga gcataaggca tctggtgttcgtatcctgtc tgctactgct 6420 attcgtaatc tggctaagat tagtaatctg ccatctactcaggcagctac tcttgcggag 6480 attcagaaat cactggcagc taaagactat aagattatctacggtacacc tactcaggtt 6540 gcagagtatg ctcgtcagaa gaatgttact gaattgacttctcaggaaat ggaagaagct 6600 caggcaggta atatttatgg ctggactaac ttcgatgataagaccattta tctggttagc 6660 ccatctatgg aaaccctcat tcatgaactg gttcatgcctctaccttcga ggaagtttat 6720 tccttctatc agggtaatga agtaagccct acttctaagcaggctattga gaaccttgaa 6780 ggtctgatgg aacagttccg ttctctggat atttccaaagattctccaga aatgagagaa 6840 gcatatgctg atgctattgc aactatcgaa ggtcatttgagtaatggatt tgttgaccca 6900 gctatctcta aagctgctgc tcttaatgag tttatggcttgggggttagc taaccgtgct 6960 cttgctgcta aacagaagag aacatcttca ctggttcaaatggtgaaaga tgtttatcag 7020 gctattaaga aattgatttg gggacgtaaa caagctcctgcattgggaga agatatgttc 7080 tccaatctgc tgtttaactc tgcaattctg atgcgtagccaacctacaac tcaggcagta 7140 gctaaagatg gcacactgtt ccatagcaaa gcatatggtaataatgaacg tctgtctcag 7200 ttgaaccaga ctttcgataa actggtaact gattaccttcgtactgaccc agttacagaa 7260 gtagaacgtc gtggcaatgt ggctaatgca ttaatgagtgctactcgact ggttcgtgat 7320 gttcagtctc atggcttcaa tatgactgct caggaacagtctgtattcca gatggttact 7380 gctgcattag caactgaagc tgcgattgac ccacatgctatggctcgtgc tcaggaactt 7440 tatacccatg taatgaaaca ccttacggta gagcatttcatggctgaccc tgatagtact 7500 aaccctgctg accgttacta tgctcaacag aaatatgacaccatctctgg tgctaatctg 7560 gttgaagtag atgccaaagg tagaaccagt ctgttacctacattcctggg tctggctatg 7620 gttaatgaag aactacgttc aatcattaaa gaaatgcctgtacctaaagc agataagaaa 7680 ttagggaatg atatagatac tctgcttacc aatgcaggtactcaggtaat ggaatctctg 7740 aaccgtcgta tggctggtga ccagaaagct actaatgttcaggacagtat tgatgctttg 7800 tcagaaacaa tcatggctgc tgctttgaaa cgagagtccttctatgatgc tgtagcaacc 7860 cctaccggta acttcattga ccgtgctaat cagtacgtaacggatagcat tgaacggtta 7920 tctgaaactg ttattgagaa ggcagataag gtaattgctaacccttctaa tatagctgct 7980 aaaggtgttg ctcatctggc taaactgact gctgctattgcatctgaaaa acagggtgaa 8040 atagtggctc agggtgttat gactgctatg aaccagggtaaagtatggca acctttccat 8100 gacttagtta atgacattgt tggccgtact aagactaatgccaatgtcta tgacttaatc 8160 aaattggtta agagccagat ttctcaagac cgtcagcaattccgtgagca tttacctaca 8220 gtcattgctg gtaagttctc tcgtaaattg actgataccgaatggtctgc aatgcatact 8280 ggtttaggta aaacagattt agctgttcta cgtgaaactatgagcatggc tgaaattaga 8340 gatttactct cttcatccaa gaaagtgaaa gatgaaatctctactctgga aaaagagatt 8400 cagaaccaag caggtagaaa ctggaatctg gttcagaagaaatctaagca actggctcaa 8460 tacatgatta tgggggaagt aggtaataac ctccttcgtaatgcccatgc tattagtcgt 8520 ttgttaggtg aacgtattac taatggtcct gtggcagatgtagctgctat tgataagctc 8580 attactttgt actctctgga attgatgaat aagtctgaccgtgacctttt gtcagaattg 8640 gctcaatcag aagtggaagg tatggagttc tccattgcttatatggttgg tcaacgtact 8700 gaagagatgc gtaaagctaa aggtgataac cgtactctgctgaatcactt taaaggctat 8760 atccctgtag agaaccagca aggtgtgaat ttgattattgctgacgataa agagtttgct 8820 aagttaaata gccaatcctt tactcgtatt ggtacttatcaggggagcac tggtttccgt 8880 actggttcta aaggttatta cttcagccca gtagctgcccgtgcccctta ctctcagggt 8940 attcttcaga acgttcgtaa tactgctggt ggtgtggatattggtactgg ctttacgtta 9000 ggcactatgg ttgctgggcg tattactgac aaaccaaccgtagagcgtat taccaaagct 9060 ctggctaaag gtgagcgtgg gcgtgaacca ctgatgccaatttataacag caaaggtcag 9120 gtagttgctt atgaacaatc cgttgaccct aatatgttgaagcacctaaa ccaagacaat 9180 cactttgcta agatggttgg tgtatggcgt ggtcgtcaggtggaagaggc taaagcacaa 9240 cgttttaatg acattctcat tgagcaatta catgctatgtatgagaaaga cattaaagac 9300 tccagtgcta ataaatctca atatgtaaac ctgttaggtaaaattgatga cccagtactg 9360 gctgatgcga ttaacctgat gaacattgag actcgtcataaggccgaaga actcttcggt 9420 aaagatgagt tatgggttcg tagggatatg ctgaatgatgcacttggcta tcgtgctgca 9480 tctattggtg atgtgtggac cggtaactct cgttggtcacctagcaccct tgatactgtt 9540 aagaagatgt tcctcggtgc attcggtaat aaggcatatcatgtagtaat gaatgctgaa 9600 aataccattc agaacttagt gaaggacgct aagacagtaattgttgttaa atctgttgta 9660 gtaccggcag ttaacttcct tgctaacatc taccagatgattggacgtgg tgttcctgtt 9720 aaagatattg ctgtgaacat tcctcgtaag acgtcagagattaatcagta tattaaatct 9780 cgtttacgtc agattgatgc ggaagcagag ctacgtgctgctgaaggtaa ccctaatctg 9840 gttcgtaaac ttaaaactga gattcaatct attactgatagtcatcgtcg tatgagtatc 9900 tggcctttga ttgaagcagg tgagttctct tctattgctgatgctggtat tagtcgtgat 9960 gacctgttag tagctgaagg taagattcat gagtacatggaaaaacttgc taataaactt 10020 ccagaaaaag tacgtaatgc tggccgttac gctcttattgctaaggacac tgctctgttc 10080 cagggtatcc agaaaacagt agagtattca gactttattgctaaagccat catctatgat 10140 gatttagtga aacgtaagaa aaaatcttct tctgaagcattaggtcaggt aactgaagag 10200 tttattaact atgacagatt gcctggtcgt ttccgtggctatatggaaag tatgggtctg 10260 atgtggttct acaactttaa aattcgttcc attaaagttgctatgagcat gattagaaac 10320 aacccagtac attctctgat tgctacagta gtacctgctcctaccatgtt tggtaacgta 10380 ggtctaccaa ttcaggacaa catgctaacc atgctggctgaaggaagact ggattactca 10440 ttaggcttcg gacaaggatt aagagcacct accctcaatccttggttcaa ccttactcac 10500 taataa 10506 2 3500 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 2 Met Ser Val PheAsp Arg Leu Ala Gly Phe Ala Asp Ser Val Thr Asn 1 5 10 15 Ala Lys GlnVal Asp Val Ser Thr Ala Thr Ala Gln Lys Lys Ala Glu 20 25 30 Gln Gly ValThr Thr Pro Leu Val Ser Pro Asp Ala Ala Tyr Gln Met 35 40 45 Gln Ala AlaArg Thr Gly Asn Val Gly Ala Asn Ala Phe Glu Pro Gly 50 55 60 Thr Val GlnSer Asp Phe Met Asn Leu Thr Pro Met Gln Ile Met Asn 65 70 75 80 Lys TyrGly Val Glu Gln Gly Leu Gln Leu Ile Asn Ala Arg Ala Asp 85 90 95 Ala GlyAsn Gln Val Phe Asn Asp Ser Val Thr Thr Arg Thr Pro Gly 100 105 110 GluGlu Leu Gly Asp Ile Ala Thr Gly Val Gly Leu Gly Phe Val Asn 115 120 125Thr Leu Gly Gly Ile Gly Ala Leu Gly Ala Gly Leu Leu Asn Asp Asp 130 135140 Ala Gly Ala Val Val Ala Gln Gln Leu Ser Lys Phe Asn Asp Ala Val 145150 155 160 His Ala Thr Gln Ser Gln Ala Leu Gln Asp Lys Arg Lys Leu PheAla 165 170 175 Ala Arg Asn Leu Met Asn Glu Val Glu Ser Glu Arg Gln TyrGln Thr 180 185 190 Asp Lys Lys Glu Gly Thr Asn Asp Ile Val Ala Ser LeuSer Lys Phe 195 200 205 Gly Arg Asp Phe Val Gly Ser Ile Glu Asn Ala AlaGln Thr Asp Ser 210 215 220 Ile Ile Ser Asp Gly Leu Ala Glu Gly Val GlySer Leu Leu Gly Ala 225 230 235 240 Gly Pro Val Leu Arg Gly Ala Ser LeuLeu Gly Lys Ala Val Val Pro 245 250 255 Ala Asn Thr Leu Arg Ser Ala AlaLeu Ala Gly Ala Ile Asp Ala Gly 260 265 270 Thr Gly Thr Gln Ser Leu AlaArg Ile Ala Ser Thr Val Gly Arg Ala 275 280 285 Ala Pro Gly Met Val GlyVal Gly Ala Met Glu Ala Gly Gly Ala Tyr 290 295 300 Gln Gln Thr Ala AspGlu Ile Met Lys Met Ser Leu Lys Asp Leu Glu 305 310 315 320 Lys Ser ProVal Tyr Gln Gln His Ile Lys Asp Gly Met Ser Pro Glu 325 330 335 Gln AlaArg Arg Gln Thr Ala Ser Glu Thr Gly Leu Thr Ala Ala Ala 340 345 350 IleGln Leu Pro Ile Ala Ala Ala Thr Gly Pro Leu Val Ser Arg Phe 355 360 365Glu Met Ala Pro Phe Arg Ala Gly Ser Leu Gly Ala Val Gly Met Asn 370 375380 Leu Ala Arg Glu Thr Val Glu Glu Gly Val Gln Gly Ala Thr Gly Gln 385390 395 400 Leu Ala Gln Asn Ile Ala Gln Gln Gln Asn Ile Asp Lys Asn GlnAsp 405 410 415 Leu Leu Lys Gly Val Gly Thr Gln Ala Gly Leu Gly Ala LeuTyr Gly 420 425 430 Phe Gly Ser Ala Gly Val Val Gln Ala Pro Ala Gly AlaAla Arg Leu 435 440 445 Ala Gly Ala Ala Thr Ala Pro Val Leu Arg Thr ThrMet Ala Gly Val 450 455 460 Lys Ala Ala Gly Ser Val Ala Gly Lys Val ValSer Pro Ile Lys Asn 465 470 475 480 Thr Leu Val Ala Arg Gly Glu Arg ValMet Lys Gln Asn Glu Glu Ala 485 490 495 Ser Pro Val Ala Asp Asp Tyr ValAla Gln Ala Ala Gln Glu Ala Met 500 505 510 Ala Gln Ala Pro Glu Ala GluVal Thr Ile Arg Asp Ala Val Glu Ala 515 520 525 Thr Asp Ala Thr Pro GluGln Lys Val Ala Ala His Gln Tyr Val Ser 530 535 540 Asp Leu Met Asn AlaThr Arg Phe Asn Pro Glu Asn Tyr Gln Glu Ala 545 550 555 560 Pro Glu HisIle Arg Asn Ala Val Ala Gly Ser Thr Asp Gln Val Gln 565 570 575 Val IleGln Lys Leu Ala Asp Leu Val Asn Thr Leu Asp Glu Ser Asn 580 585 590 ProGln Ala Leu Met Glu Ala Ala Ser Tyr Met Tyr Asp Ala Val Ser 595 600 605Glu Phe Glu Gln Phe Ile Asn Arg Asp Pro Ala Ala Leu Asp Ser Ile 610 615620 Pro Lys Asp Ser Pro Ala Ile Glu Leu Leu Asn Arg Tyr Thr Asn Leu 625630 635 640 Thr Ala Asn Ile Gln Asn Thr Pro Lys Val Ile Gly Ala Leu AsnVal 645 650 655 Ile Asn Arg Met Ile Asn Glu Ser Ala Gln Asn Gly Ser LeuAsn Val 660 665 670 Thr Glu Glu Ser Ser Pro Gln Glu Met Gln Asn Val AlaLeu Ala Ala 675 680 685 Glu Val Ala Pro Glu Lys Leu Asn Pro Glu Ser ValAsn Val Val Leu 690 695 700 Lys His Ala Ala Asp Gly Arg Ile Lys Leu AsnAsn Arg Gln Ile Ala 705 710 715 720 Ala Leu Gln Asn Ala Ala Ala Ile LeuLys Gly Ala Arg Glu Tyr Asp 725 730 735 Ala Glu Ala Ala Arg Leu Gly LeuArg Pro Gln Asp Ile Val Ser Lys 740 745 750 Gln Ile Lys Thr Asp Glu SerArg Thr Gln Glu Gly Gln Tyr Ser Ala 755 760 765 Leu Gln His Ala Asn ArgIle Arg Ser Ala Tyr Asn Ser Gly Asn Phe 770 775 780 Glu Leu Ala Ser AlaTyr Leu Asn Asp Phe Met Gln Phe Ala Gln His 785 790 795 800 Met Gln AsnLys Val Gly Ala Leu Asn Glu His Leu Val Thr Gly Asn 805 810 815 Ala AspLys Asn Lys Ser Val His Tyr Gln Ala Leu Thr Ala Asp Arg 820 825 830 GluTrp Val Arg Ser Arg Thr Gly Leu Gly Val Asn Pro Tyr Asp Thr 835 840 845Lys Ser Val Lys Phe Ala Gln Gln Val Ala Leu Glu Ala Lys Thr Val 850 855860 Ala Asp Ile Ala Asn Ala Leu Ala Ser Ala Tyr Pro Glu Leu Lys Val 865870 875 880 Ser His Ile Lys Val Thr Pro Leu Asp Ser Arg Leu Asn Ala ProAla 885 890 895 Ala Glu Val Val Lys Ala Phe Arg Gln Gly Asn Arg Asp ValAla Ser 900 905 910 Ser Gln Pro Lys Ala Asp Ser Val Asn Gln Val Lys GluThr Pro Val 915 920 925 Thr Lys Gln Glu Pro Val Thr Ser Thr Val Gln ThrLys Thr Pro Val 930 935 940 Ser Glu Ser Val Lys Thr Glu Pro Thr Thr LysGlu Ser Ser Pro Gln 945 950 955 960 Ala Ile Lys Glu Pro Val Asn Gln SerGlu Lys Gln Asp Val Asn Leu 965 970 975 Thr Asn Glu Asp Asn Ile Lys GlnPro Thr Glu Ser Val Lys Glu Thr 980 985 990 Glu Thr Ser Thr Lys Glu SerThr Val Thr Glu Glu Leu Lys Glu Gly 995 1000 1005 Ile Asp Ala Val TyrPro Ser Leu Val Gly Thr Ala Asp Ser Lys Ala 1010 1015 1020 Glu Gly IleLys Asn Tyr Phe Lys Leu Ser Phe Thr Leu Pro Glu Glu 1025 1030 1035 1040Gln Lys Ser Arg Thr Val Gly Ser Glu Ala Pro Leu Lys Asp Val Ala 10451050 1055 Gln Ala Leu Ser Ser Arg Ala Arg Tyr Glu Leu Phe Thr Glu LysGlu 1060 1065 1070 Thr Ala Asn Pro Ala Phe Asn Gly Glu Val Ile Lys ArgTyr Lys Glu 1075 1080 1085 Leu Met Glu His Gly Glu Gly Ile Ala Asp IleLeu Arg Ser Arg Leu 1090 1095 1100 Ala Lys Phe Leu Asn Thr Lys Asp ValGly Lys Arg Phe Ala Gln Gly 1105 1110 1115 1120 Thr Glu Ala Asn Arg TrpVal Gly Gly Lys Leu Leu Asn Ile Val Glu 1125 1130 1135 Gln Asp Gly AspThr Phe Lys Tyr Asn Glu Gln Leu Leu Gln Thr Ala 1140 1145 1150 Val LeuAla Gly Leu Gln Trp Arg Leu Thr Ala Thr Ser Asn Thr Ala 1155 1160 1165Ile Lys Asp Ala Lys Asp Val Ala Ala Ile Thr Gly Ile Asp Gln Ala 11701175 1180 Leu Leu Pro Glu Gly Leu Val Glu Gln Phe Asp Thr Gly Met ThrLeu 1185 1190 1195 1200 Thr Glu Ala Val Ser Ser Leu Ala Gln Lys Ile GluSer Tyr Trp Gly 1205 1210 1215 Leu Ser Arg Asn Pro Asn Ala Pro Leu GlyTyr Thr Lys Gly Ile Pro 1220 1225 1230 Thr Ala Met Ala Ala Glu Ile LeuAla Ala Phe Val Glu Ser Thr Asp 1235 1240 1245 Val Val Glu Asn Ile ValAsp Met Ser Glu Ile Asp Pro Asp Asn Lys 1250 1255 1260 Lys Thr Ile GlyLeu Tyr Thr Ile Thr Glu Leu Asp Ser Phe Asp Pro 1265 1270 1275 1280 IleAsn Ser Phe Pro Thr Ala Ile Glu Glu Ala Val Leu Val Asn Pro 1285 12901295 Thr Glu Lys Met Phe Phe Gly Asp Asp Ile Pro Pro Val Ala Asn Thr1300 1305 1310 Gln Leu Arg Asn Pro Ala Val Arg Asn Thr Pro Glu Gln LysAla Ala 1315 1320 1325 Leu Lys Ala Glu Gln Ala Thr Glu Phe Tyr Val HisThr Pro Met Val 1330 1335 1340 Gln Phe Tyr Glu Thr Leu Gly Lys Asp ArgIle Leu Glu Leu Met Gly 1345 1350 1355 1360 Ala Gly Thr Leu Asn Lys GluLeu Leu Asn Asp Asn His Ala Lys Ser 1365 1370 1375 Leu Glu Gly Lys AsnArg Ser Val Glu Asp Ser Tyr Asn Gln Leu Phe 1380 1385 1390 Ser Val IleGlu Gln Val Arg Ala Gln Ser Glu Asp Ile Ser Thr Val 1395 1400 1405 ProIle His Tyr Ala Tyr Asn Met Thr Arg Val Gly Arg Met Gln Met 1410 14151420 Leu Gly Lys Tyr Asn Pro Gln Ser Ala Lys Leu Val Arg Glu Ala Ile1425 1430 1435 1440 Leu Pro Thr Lys Ala Thr Leu Asp Leu Ser Asn Gln AsnAsn Glu Asp 1445 1450 1455 Phe Ser Ala Phe Gln Leu Gly Leu Ala Gln AlaLeu Asp Ile Lys Val 1460 1465 1470 His Thr Met Thr Arg Glu Val Met SerAsp Glu Leu Thr Lys Leu Leu 1475 1480 1485 Glu Gly Asn Leu Lys Pro AlaIle Asp Met Met Val Glu Phe Asn Thr 1490 1495 1500 Thr Gly Ser Leu ProGlu Asn Ala Val Asp Val Leu Asn Thr Ala Leu 1505 1510 1515 1520 Gly AspArg Lys Ser Phe Val Ala Leu Met Ala Leu Met Glu Tyr Ser 1525 1530 1535Arg Tyr Leu Val Ala Glu Asp Lys Ser Ala Phe Val Thr Pro Leu Tyr 15401545 1550 Val Glu Ala Asp Gly Val Thr Asn Gly Pro Ile Asn Ala Met MetLeu 1555 1560 1565 Met Thr Gly Gly Leu Phe Thr Pro Asp Trp Ile Arg AsnIle Ala Lys 1570 1575 1580 Gly Gly Leu Phe Ile Gly Ser Pro Asn Lys ThrMet Asn Glu His Arg 1585 1590 1595 1600 Ser Thr Ala Asp Asn Asn Asp LeuTyr Gln Ala Ser Thr Asn Ala Leu 1605 1610 1615 Met Glu Ser Leu Gly LysLeu Arg Ser Asn Tyr Ala Ser Asn Met Pro 1620 1625 1630 Ile Gln Ser GlnIle Asp Ser Leu Leu Ser Leu Met Asp Leu Phe Leu 1635 1640 1645 Pro AspIle Asn Leu Gly Glu Asn Gly Ala Leu Glu Leu Lys Arg Gly 1650 1655 1660Ile Ala Lys Asn Pro Leu Thr Ile Thr Ile Tyr Gly Ser Gly Ala Arg 16651670 1675 1680 Gly Ile Ala Gly Lys Leu Val Ser Ser Val Thr Asp Ala IleTyr Glu 1685 1690 1695 Arg Met Ser Asp Val Leu Lys Ala Arg Ala Lys AspPro Asn Ile Ser 1700 1705 1710 Ala Ala Met Ala Met Phe Gly Lys Gln AlaAla Ser Glu Ala His Ala 1715 1720 1725 Glu Glu Leu Leu Ala Arg Phe LeuLys Asp Met Glu Thr Leu Thr Ser 1730 1735 1740 Thr Val Pro Val Lys ArgLys Gly Val Leu Glu Leu Gln Ser Thr Gly 1745 1750 1755 1760 Thr Gly AlaLys Gly Lys Ile Asn Pro Lys Thr Tyr Thr Ile Lys Gly 1765 1770 1775 GluGln Leu Lys Ala Leu Gln Glu Asn Met Leu His Phe Phe Val Glu 1780 17851790 Pro Leu Arg Asn Gly Ile Thr Gln Thr Val Gly Glu Ser Leu Val Tyr1795 1800 1805 Ser Thr Glu Gln Leu Gln Lys Ala Thr Gln Ile Gln Ser ValVal Leu 1810 1815 1820 Glu Asp Met Phe Lys Gln Arg Val Gln Glu Lys LeuAla Glu Lys Ala 1825 1830 1835 1840 Lys Asp Pro Thr Trp Lys Lys Gly AspPhe Leu Thr Gln Lys Glu Leu 1845 1850 1855 Asn Asp Ile Gln Ala Ser LeuAsn Asn Leu Ala Pro Met Ile Glu Thr 1860 1865 1870 Gly Ser Gln Thr PheTyr Ile Ala Gly Ser Glu Asn Ala Glu Val Ala 1875 1880 1885 Asn Gln ValLeu Ala Thr Asn Leu Asp Asp Arg Met Arg Val Pro Met 1890 1895 1900 SerIle Tyr Ala Pro Ala Gln Ala Gly Val Ala Gly Ile Pro Phe Met 1905 19101915 1920 Thr Ile Gly Thr Gly Asp Gly Met Met Met Gln Thr Leu Ser ThrMet 1925 1930 1935 Lys Gly Ala Pro Lys Asn Thr Leu Lys Ile Phe Asp GlyMet Asn Ile 1940 1945 1950 Gly Leu Asn Asp Ile Thr Asp Ala Ser Arg LysAla Asn Glu Ala Val 1955 1960 1965 Tyr Thr Ser Trp Gln Gly Asn Pro IleLys Asn Val Tyr Glu Ser Tyr 1970 1975 1980 Ala Lys Phe Met Lys Asn ValAsp Phe Ser Lys Leu Ser Pro Glu Ala 1985 1990 1995 2000 Leu Glu Ala IleGly Lys Ser Ala Leu Glu Tyr Asp Gln Arg Glu Asn 2005 2010 2015 Ala ThrVal Asp Asp Ile Ala Asn Ala Ala Ser Leu Ile Glu Arg Asn 2020 2025 2030Leu Arg Asn Ile Ala Leu Gly Val Asp Ile Arg His Lys Val Leu Asp 20352040 2045 Lys Val Asn Leu Ser Ile Asp Gln Met Ala Ala Val Gly Ala ProTyr 2050 2055 2060 Gln Asn Asn Gly Lys Ile Asp Leu Ser Asn Met Thr ProGlu Gln Gln 2065 2070 2075 2080 Ala Asp Glu Leu Asn Lys Leu Phe Arg GluGlu Leu Glu Ala Arg Lys 2085 2090 2095 Gln Lys Val Ala Lys Ala Arg AlaGlu Val Lys Glu Glu Thr Val Ser 2100 2105 2110 Glu Lys Glu Pro Val AsnPro Asp Phe Gly Met Val Gly Arg Glu His 2115 2120 2125 Lys Ala Ser GlyVal Arg Ile Leu Ser Ala Thr Ala Ile Arg Asn Leu 2130 2135 2140 Ala LysIle Ser Asn Leu Pro Ser Thr Gln Ala Ala Thr Leu Ala Glu 2145 2150 21552160 Ile Gln Lys Ser Leu Ala Ala Lys Asp Tyr Lys Ile Ile Tyr Gly Thr2165 2170 2175 Pro Thr Gln Val Ala Glu Tyr Ala Arg Gln Lys Asn Val ThrGlu Leu 2180 2185 2190 Thr Ser Gln Glu Met Glu Glu Ala Gln Ala Gly AsnIle Tyr Gly Trp 2195 2200 2205 Thr Asn Phe Asp Asp Lys Thr Ile Tyr LeuVal Ser Pro Ser Met Glu 2210 2215 2220 Thr Leu Ile His Glu Leu Val HisAla Ser Thr Phe Glu Glu Val Tyr 2225 2230 2235 2240 Ser Phe Tyr Gln GlyAsn Glu Val Ser Pro Thr Ser Lys Gln Ala Ile 2245 2250 2255 Glu Asn LeuGlu Gly Leu Met Glu Gln Phe Arg Ser Leu Asp Ile Ser 2260 2265 2270 LysAsp Ser Pro Glu Met Arg Glu Ala Tyr Ala Asp Ala Ile Ala Thr 2275 22802285 Ile Glu Gly His Leu Ser Asn Gly Phe Val Asp Pro Ala Ile Ser Lys2290 2295 2300 Ala Ala Ala Leu Asn Glu Phe Met Ala Trp Gly Leu Ala AsnArg Ala 2305 2310 2315 2320 Leu Ala Ala Lys Gln Lys Arg Thr Ser Ser LeuVal Gln Met Val Lys 2325 2330 2335 Asp Val Tyr Gln Ala Ile Lys Lys LeuIle Trp Gly Arg Lys Gln Ala 2340 2345 2350 Pro Ala Leu Gly Glu Asp MetPhe Ser Asn Leu Leu Phe Asn Ser Ala 2355 2360 2365 Ile Leu Met Arg SerGln Pro Thr Thr Gln Ala Val Ala Lys Asp Gly 2370 2375 2380 Thr Leu PheHis Ser Lys Ala Tyr Gly Asn Asn Glu Arg Leu Ser Gln 2385 2390 2395 2400Leu Asn Gln Thr Phe Asp Lys Leu Val Thr Asp Tyr Leu Arg Thr Asp 24052410 2415 Pro Val Thr Glu Val Glu Arg Arg Gly Asn Val Ala Asn Ala LeuMet 2420 2425 2430 Ser Ala Thr Arg Leu Val Arg Asp Val Gln Ser His GlyPhe Asn Met 2435 2440 2445 Thr Ala Gln Glu Gln Ser Val Phe Gln Met ValThr Ala Ala Leu Ala 2450 2455 2460 Thr Glu Ala Ala Ile Asp Pro His AlaMet Ala Arg Ala Gln Glu Leu 2465 2470 2475 2480 Tyr Thr His Val Met LysHis Leu Thr Val Glu His Phe Met Ala Asp 2485 2490 2495 Pro Asp Ser ThrAsn Pro Ala Asp Arg Tyr Tyr Ala Gln Gln Lys Tyr 2500 2505 2510 Asp ThrIle Ser Gly Ala Asn Leu Val Glu Val Asp Ala Lys Gly Arg 2515 2520 2525Thr Ser Leu Leu Pro Thr Phe Leu Gly Leu Ala Met Val Asn Glu Glu 25302535 2540 Leu Arg Ser Ile Ile Lys Glu Met Pro Val Pro Lys Ala Asp LysLys 2545 2550 2555 2560 Leu Gly Asn Asp Ile Asp Thr Leu Leu Thr Asn AlaGly Thr Gln Val 2565 2570 2575 Met Glu Ser Leu Asn Arg Arg Met Ala GlyAsp Gln Lys Ala Thr Asn 2580 2585 2590 Val Gln Asp Ser Ile Asp Ala LeuSer Glu Thr Ile Met Ala Ala Ala 2595 2600 2605 Leu Lys Arg Glu Ser PheTyr Asp Ala Val Ala Thr Pro Thr Gly Asn 2610 2615 2620 Phe Ile Asp ArgAla Asn Gln Tyr Val Thr Asp Ser Ile Glu Arg Leu 2625 2630 2635 2640 SerGlu Thr Val Ile Glu Lys Ala Asp Lys Val Ile Ala Asn Pro Ser 2645 26502655 Asn Ile Ala Ala Lys Gly Val Ala His Leu Ala Lys Leu Thr Ala Ala2660 2665 2670 Ile Ala Ser Glu Lys Gln Gly Glu Ile Val Ala Gln Gly ValMet Thr 2675 2680 2685 Ala Met Asn Gln Gly Lys Val Trp Gln Pro Phe HisAsp Leu Val Asn 2690 2695 2700 Asp Ile Val Gly Arg Thr Lys Thr Asn AlaAsn Val Tyr Asp Leu Ile 2705 2710 2715 2720 Lys Leu Val Lys Ser Gln IleSer Gln Asp Arg Gln Gln Phe Arg Glu 2725 2730 2735 His Leu Pro Thr ValIle Ala Gly Lys Phe Ser Arg Lys Leu Thr Asp 2740 2745 2750 Thr Glu TrpSer Ala Met His Thr Gly Leu Gly Lys Thr Asp Leu Ala 2755 2760 2765 ValLeu Arg Glu Thr Met Ser Met Ala Glu Ile Arg Asp Leu Leu Ser 2770 27752780 Ser Ser Lys Lys Val Lys Asp Glu Ile Ser Thr Leu Glu Lys Glu Ile2785 2790 2795 2800 Gln Asn Gln Ala Gly Arg Asn Trp Asn Leu Val Gln LysLys Ser Lys 2805 2810 2815 Gln Leu Ala Gln Tyr Met Ile Met Gly Glu ValGly Asn Asn Leu Leu 2820 2825 2830 Arg Asn Ala His Ala Ile Ser Arg LeuLeu Gly Glu Arg Ile Thr Asn 2835 2840 2845 Gly Pro Val Ala Asp Val AlaAla Ile Asp Lys Leu Ile Thr Leu Tyr 2850 2855 2860 Ser Leu Glu Leu MetAsn Lys Ser Asp Arg Asp Leu Leu Ser Glu Leu 2865 2870 2875 2880 Ala GlnSer Glu Val Glu Gly Met Glu Phe Ser Ile Ala Tyr Met Val 2885 2890 2895Gly Gln Arg Thr Glu Glu Met Arg Lys Ala Lys Gly Asp Asn Arg Thr 29002905 2910 Leu Leu Asn His Phe Lys Gly Tyr Ile Pro Val Glu Asn Gln GlnGly 2915 2920 2925 Val Asn Leu Ile Ile Ala Asp Asp Lys Glu Phe Ala LysLeu Asn Ser 2930 2935 2940 Gln Ser Phe Thr Arg Ile Gly Thr Tyr Gln GlySer Thr Gly Phe Arg 2945 2950 2955 2960 Thr Gly Ser Lys Gly Tyr Tyr PheSer Pro Val Ala Ala Arg Ala Pro 2965 2970 2975 Tyr Ser Gln Gly Ile LeuGln Asn Val Arg Asn Thr Ala Gly Gly Val 2980 2985 2990 Asp Ile Gly ThrGly Phe Thr Leu Gly Thr Met Val Ala Gly Arg Ile 2995 3000 3005 Thr AspLys Pro Thr Val Glu Arg Ile Thr Lys Ala Leu Ala Lys Gly 3010 3015 3020Glu Arg Gly Arg Glu Pro Leu Met Pro Ile Tyr Asn Ser Lys Gly Gln 30253030 3035 3040 Val Val Ala Tyr Glu Gln Ser Val Asp Pro Asn Met Leu LysHis Leu 3045 3050 3055 Asn Gln Asp Asn His Phe Ala Lys Met Val Gly ValTrp Arg Gly Arg 3060 3065 3070 Gln Val Glu Glu Ala Lys Ala Gln Arg PheAsn Asp Ile Leu Ile Glu 3075 3080 3085 Gln Leu His Ala Met Tyr Glu LysAsp Ile Lys Asp Ser Ser Ala Asn 3090 3095 3100 Lys Ser Gln Tyr Val AsnLeu Leu Gly Lys Ile Asp Asp Pro Val Leu 3105 3110 3115 3120 Ala Asp AlaIle Asn Leu Met Asn Ile Glu Thr Arg His Lys Ala Glu 3125 3130 3135 GluLeu Phe Gly Lys Asp Glu Leu Trp Val Arg Arg Asp Met Leu Asn 3140 31453150 Asp Ala Leu Gly Tyr Arg Ala Ala Ser Ile Gly Asp Val Trp Thr Gly3155 3160 3165 Asn Ser Arg Trp Ser Pro Ser Thr Leu Asp Thr Val Lys LysMet Phe 3170 3175 3180 Leu Gly Ala Phe Gly Asn Lys Ala Tyr His Val ValMet Asn Ala Glu 3185 3190 3195 3200 Asn Thr Ile Gln Asn Leu Val Lys AspAla Lys Thr Val Ile Val Val 3205 3210 3215 Lys Ser Val Val Val Pro AlaVal Asn Phe Leu Ala Asn Ile Tyr Gln 3220 3225 3230 Met Ile Gly Arg GlyVal Pro Val Lys Asp Ile Ala Val Asn Ile Pro 3235 3240 3245 Arg Lys ThrSer Glu Ile Asn Gln Tyr Ile Lys Ser Arg Leu Arg Gln 3250 3255 3260 IleAsp Ala Glu Ala Glu Leu Arg Ala Ala Glu Gly Asn Pro Asn Leu 3265 32703275 3280 Val Arg Lys Leu Lys Thr Glu Ile Gln Ser Ile Thr Asp Ser HisArg 3285 3290 3295 Arg Met Ser Ile Trp Pro Leu Ile Glu Ala Gly Glu PheSer Ser Ile 3300 3305 3310 Ala Asp Ala Gly Ile Ser Arg Asp Asp Leu LeuVal Ala Glu Gly Lys 3315 3320 3325 Ile His Glu Tyr Met Glu Lys Leu AlaAsn Lys Leu Pro Glu Lys Val 3330 3335 3340 Arg Asn Ala Gly Arg Tyr AlaLeu Ile Ala Lys Asp Thr Ala Leu Phe 3345 3350 3355 3360 Gln Gly Ile GlnLys Thr Val Glu Tyr Ser Asp Phe Ile Ala Lys Ala 3365 3370 3375 Ile IleTyr Asp Asp Leu Val Lys Arg Lys Lys Lys Ser Ser Ser Glu 3380 3385 3390Ala Leu Gly Gln Val Thr Glu Glu Phe Ile Asn Tyr Asp Arg Leu Pro 33953400 3405 Gly Arg Phe Arg Gly Tyr Met Glu Ser Met Gly Leu Met Trp PheTyr 3410 3415 3420 Asn Phe Lys Ile Arg Ser Ile Lys Val Ala Met Ser MetIle Arg Asn 3425 3430 3435 3440 Asn Pro Val His Ser Leu Ile Ala Thr ValVal Pro Ala Pro Thr Met 3445 3450 3455 Phe Gly Asn Val Gly Leu Pro IleGln Asp Asn Met Leu Thr Met Leu 3460 3465 3470 Ala Glu Gly Arg Leu AspTyr Ser Leu Gly Phe Gly Gln Gly Leu Arg 3475 3480 3485 Ala Pro Thr LeuAsn Pro Trp Phe Asn Leu Thr His 3490 3495 3500 3 3318 DNA ArtificialSequence Description of Artificial Sequence Synthetic Primer 3gaaagtacag ttacagaaga attaaaagaa ggtattgatg ctgtttaccc ttcattggta 60ggtactgctg attctaaagc agagggtatt aagaactatt tcaaattgtc ctttacctta 120ccagaagaac agaaatcccg tactgttggt tcagaagcac ctctaaaaga tgtagcccaa 180gctctgtctt ctcgtgctcg ttatgaactc tttactgaga aagaaactgc taaccctgct 240tttaatgggg aagttattaa gcgatacaaa gaactcatgg aacatgggga aggtattgct 300gatattcttc gctcccgtct ggctaagttc cttaacacta aggatgttgg taaacgtttt 360gctcaaggta cagaagccaa ccgttgggta ggtggtaagt tacttaacat tgttgagcag 420gatggggata cctttaagta caacgaacaa ttgctacaga ctgctgtatt agcaggtctt 480caatggagac ttactgctac cagcaatact gctatcaaag atgcaaaaga tgttgctgct 540attactggta ttgaccaagc tctgctgcca gaaggtttag tagagcaatt tgatactggt 600atgacactca ctgaagcagt tagttccctg gctcagaaaa ttgagtctta ctggggatta 660tctcgtaatc caaatgctcc attgggctat accaaaggca tccctacagc aatggctgct 720gaaattctgg ctgcatttgt agagtctact gatgttgtag agaacatcgt ggatatgtca 780gaaattgacc cagataacaa gaagactatt ggtctgtaca ccattactga actggattcc 840ttcgacccaa ttaatagctt ccctactgct attgaagaag ctgttttagt gaatcctaca 900gagaagatgt tctttggtga tgacattcct cctgtagcta atactcagct tcgtaaccct 960gctgttcgta atactccaga acagaaggct gcattgaaag cagagcaggc tacagagttc 1020tatgtacaca ccccaatggt tcaattctat gagacgttag gtaaagaccg tattctcgaa 1080ctgatgggtg ctggtactct gaataaagag ttacttaatg ataaccatgc taaatctctg 1140gaaggtaaga accgttcagt agaggactct tacaaccaac tgttctccgt cattgagcag 1200gtaagagcac agagcgaaga catctctact gtacctattc actatgcata caatatgacc 1260cgtgttggtc gtatgcagat gttaggtaaa tacaatcctc aatcagccaa actggttcgt 1320gaggccatct tacctactaa agctactttg gatttatcga accagaacaa tgaagacttc 1380tctgcattcc agttaggtct ggctcaggca ttggacatta aagtccatac tatgactcgt 1440gaggttatgt ctgacgagtt gactaaatta ctggaaggta atctgaaacc agccattgat 1500atgatggttg agtttaatac cactggttcc ttaccagaaa acgcagttga tgttctgaat 1560acagcattag gagataggaa gtcattcgta gcattgatgg ctcttatgga gtattcccgt 1620tacttagtag cagaggataa atctgcattt gtaactccac tgtatgtaga agcagatggt 1680gttactaatg gtccaatcaa tgccatgatg ctaatgacag gcggtctgtt tactcctgac 1740tggattcgta atattgccaa agggggcttg ttcattggtt ctccaaataa gaccatgaat 1800gagcatcgct ctactgctga caataatgat ttatatcaag catccactaa tgctttgatg 1860gaatcgttgg gtaagttacg tagtaactat gcctctaata tgcctattca gtctcagata 1920gacagtcttc tttctctgat ggatttgttt ttaccggata ttaatcttgg tgagaatggt 1980gctttagaac ttaaacgtgg tattgctaag aacccactga ctattaccat ctatggttct 2040ggtgctcgtg gtattgcagg taagctggtt agttctgtta ctgatgccat ctatgagcgt 2100atgtctgatg tactgaaagc tcgtgctaaa gacccaaata tctctgctgc tatggcaatg 2160tttggtaagc aagctgcttc agaagcacat gctgaagaac ttcttgcccg tttcctgaaa 2220gatatggaaa cactgacttc tactgttcct gttaaacgta aaggtgtact ggaactacaa 2280tccacaggta caggagccaa aggaaaaatc aatcctaaga cctataccat taagggcgag 2340caactgaagg cacttcagga aaatatgctg cacttctttg tagaaccact acgtaatggt 2400attactcaga ctgtaggtga aagtctggtg tactctactg aacaattaca gaaagctact 2460cagattcaat ctgtagtgct ggaagatatg ttcaaacagc gagtacaaga gaagctggca 2520gagaaggcta aagacccaac atggaagaaa ggtgatttcc ttactcagaa agaactgaat 2580gatattcagg cttctctgaa taacttagcc cctatgattg agactggttc tcagactttc 2640tacattgctg gttcagaaaa tgcagaagta gcaaatcagg tattagctac taaccttgat 2700gaccgtatgc gtgtaccaat gagtatctat gctccagcac aggccggtgt agcaggtatt 2760ccatttatga ctattggtac tggtgatggc atgatgatgc aaactctttc cactatgaaa 2820ggtgcaccaa agaataccct caaaatcttt gatggtatga acattggttt gaatgacatc 2880actgatgcca gtcgtaaagc taatgaagct gtttacactt cttggcaggg taaccctatt 2940aagaatgttt atgaatcata tgctaagttc atgaagaatg tagatttcag caagctgtcc 3000cctgaagcat tggaagcaat tggtaaatct gctctggaat atgaccaacg tgagaatgct 3060actgtagatg atattgctaa cgctgcatct ctgattgaac gtaacttacg taatattgca 3120ctgggtgtag atattcgtca taaggtgctg gataaggtaa atctgtccat tgaccagatg 3180gctgctgtag gtgctcctta tcagaacaac ggtaagattg acctcagcaa tatgacccct 3240gaacaacagg ctgatgaact gaataaactt ttccgtgaag agttagaagc ccgtaaacaa 3300aaagtcgcta aggctagg 3318 4 1107 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 4 Met Glu Ser Thr Val Thr Glu GluLeu Lys Glu Gly Ile Asp Ala Val 1 5 10 15 Tyr Pro Ser Leu Val Gly ThrAla Asp Ser Lys Ala Glu Gly Ile Lys 20 25 30 Asn Tyr Phe Lys Leu Ser PheThr Leu Pro Glu Glu Gln Lys Ser Arg 35 40 45 Thr Val Gly Ser Glu Ala ProLeu Lys Asp Val Ala Gln Ala Leu Ser 50 55 60 Ser Arg Ala Arg Tyr Glu LeuPhe Thr Glu Lys Glu Thr Ala Asn Pro 65 70 75 80 Ala Phe Asn Gly Glu ValIle Lys Arg Tyr Lys Glu Leu Met Glu His 85 90 95 Gly Glu Gly Ile Ala AspIle Leu Arg Ser Arg Leu Ala Lys Phe Leu 100 105 110 Asn Thr Lys Asp ValGly Lys Arg Phe Ala Gln Gly Thr Glu Ala Asn 115 120 125 Arg Trp Val GlyGly Lys Leu Leu Asn Ile Val Glu Gln Asp Gly Asp 130 135 140 Thr Phe LysTyr Asn Glu Gln Leu Leu Gln Thr Ala Val Leu Ala Gly 145 150 155 160 LeuGln Trp Arg Leu Thr Ala Thr Ser Asn Thr Ala Ile Lys Asp Ala 165 170 175Lys Asp Val Ala Ala Ile Thr Gly Ile Asp Gln Ala Leu Leu Pro Glu 180 185190 Gly Leu Val Glu Gln Phe Asp Thr Gly Met Thr Leu Thr Glu Ala Val 195200 205 Ser Ser Leu Ala Gln Lys Ile Glu Ser Tyr Trp Gly Leu Ser Arg Asn210 215 220 Pro Asn Ala Pro Leu Gly Tyr Thr Lys Gly Ile Pro Thr Ala MetAla 225 230 235 240 Ala Glu Ile Leu Ala Ala Phe Val Glu Ser Thr Asp ValVal Glu Asn 245 250 255 Ile Val Asp Met Ser Glu Ile Asp Pro Asp Asn LysLys Thr Ile Gly 260 265 270 Leu Tyr Thr Ile Thr Glu Leu Asp Ser Phe AspPro Ile Asn Ser Phe 275 280 285 Pro Thr Ala Ile Glu Glu Ala Val Leu ValAsn Pro Thr Glu Lys Met 290 295 300 Phe Phe Gly Asp Asp Ile Pro Pro ValAla Asn Thr Gln Leu Arg Asn 305 310 315 320 Pro Ala Val Arg Asn Thr ProGlu Gln Lys Ala Ala Leu Lys Ala Glu 325 330 335 Gln Ala Thr Glu Phe TyrVal His Thr Pro Met Val Gln Phe Tyr Glu 340 345 350 Thr Leu Gly Lys AspArg Ile Leu Glu Leu Met Gly Ala Gly Thr Leu 355 360 365 Asn Lys Glu LeuLeu Asn Asp Asn His Ala Lys Ser Leu Glu Gly Lys 370 375 380 Asn Arg SerVal Glu Asp Ser Tyr Asn Gln Leu Phe Ser Val Ile Glu 385 390 395 400 GlnVal Arg Ala Gln Ser Glu Asp Ile Ser Thr Val Pro Ile His Tyr 405 410 415Ala Tyr Asn Met Thr Arg Val Gly Arg Met Gln Met Leu Gly Lys Tyr 420 425430 Asn Pro Gln Ser Ala Lys Leu Val Arg Glu Ala Ile Leu Pro Thr Lys 435440 445 Ala Thr Leu Asp Leu Ser Asn Gln Asn Asn Glu Asp Phe Ser Ala Phe450 455 460 Gln Leu Gly Leu Ala Gln Ala Leu Asp Ile Lys Val His Thr MetThr 465 470 475 480 Arg Glu Val Met Ser Asp Glu Leu Thr Lys Leu Leu GluGly Asn Leu 485 490 495 Lys Pro Ala Ile Asp Met Met Val Glu Phe Asn ThrThr Gly Ser Leu 500 505 510 Pro Glu Asn Ala Val Asp Val Leu Asn Thr AlaLeu Gly Asp Arg Lys 515 520 525 Ser Phe Val Ala Leu Met Ala Leu Met GluTyr Ser Arg Tyr Leu Val 530 535 540 Ala Glu Asp Lys Ser Ala Phe Val ThrPro Leu Tyr Val Glu Ala Asp 545 550 555 560 Gly Val Thr Asn Gly Pro IleAsn Ala Met Met Leu Met Thr Gly Gly 565 570 575 Leu Phe Thr Pro Asp TrpIle Arg Asn Ile Ala Lys Gly Gly Leu Phe 580 585 590 Ile Gly Ser Pro AsnLys Thr Met Asn Glu His Arg Ser Thr Ala Asp 595 600 605 Asn Asn Asp LeuTyr Gln Ala Ser Thr Asn Ala Leu Met Glu Ser Leu 610 615 620 Gly Lys LeuArg Ser Asn Tyr Ala Ser Asn Met Pro Ile Gln Ser Gln 625 630 635 640 IleAsp Ser Leu Leu Ser Leu Met Asp Leu Phe Leu Pro Asp Ile Asn 645 650 655Leu Gly Glu Asn Gly Ala Leu Glu Leu Lys Arg Gly Ile Ala Lys Asn 660 665670 Pro Leu Thr Ile Thr Ile Tyr Gly Ser Gly Ala Arg Gly Ile Ala Gly 675680 685 Lys Leu Val Ser Ser Val Thr Asp Ala Ile Tyr Glu Arg Met Ser Asp690 695 700 Val Leu Lys Ala Arg Ala Lys Asp Pro Asn Ile Ser Ala Ala MetAla 705 710 715 720 Met Phe Gly Lys Gln Ala Ala Ser Glu Ala His Ala GluGlu Leu Leu 725 730 735 Ala Arg Phe Leu Lys Asp Met Glu Thr Leu Thr SerThr Val Pro Val 740 745 750 Lys Arg Lys Gly Val Leu Glu Leu Gln Ser ThrGly Thr Gly Ala Lys 755 760 765 Gly Lys Ile Asn Pro Lys Thr Tyr Thr IleLys Gly Glu Gln Leu Lys 770 775 780 Ala Leu Gln Glu Asn Met Leu His PhePhe Val Glu Pro Leu Arg Asn 785 790 795 800 Gly Ile Thr Gln Thr Val GlyGlu Ser Leu Val Tyr Ser Thr Glu Gln 805 810 815 Leu Gln Lys Ala Thr GlnIle Gln Ser Val Val Leu Glu Asp Met Phe 820 825 830 Lys Gln Arg Val GlnGlu Lys Leu Ala Glu Lys Ala Lys Asp Pro Thr 835 840 845 Trp Lys Lys GlyAsp Phe Leu Thr Gln Lys Glu Leu Asn Asp Ile Gln 850 855 860 Ala Ser LeuAsn Asn Leu Ala Pro Met Ile Glu Thr Gly Ser Gln Thr 865 870 875 880 PheTyr Ile Ala Gly Ser Glu Asn Ala Glu Val Ala Asn Gln Val Leu 885 890 895Ala Thr Asn Leu Asp Asp Arg Met Arg Val Pro Met Ser Ile Tyr Ala 900 905910 Pro Ala Gln Ala Gly Val Ala Gly Ile Pro Phe Met Thr Ile Gly Thr 915920 925 Gly Asp Gly Met Met Met Gln Thr Leu Ser Thr Met Lys Gly Ala Pro930 935 940 Lys Asn Thr Leu Lys Ile Phe Asp Gly Met Asn Ile Gly Leu AsnAsp 945 950 955 960 Ile Thr Asp Ala Ser Arg Lys Ala Asn Glu Ala Val TyrThr Ser Trp 965 970 975 Gln Gly Asn Pro Ile Lys Asn Val Tyr Glu Ser TyrAla Lys Phe Met 980 985 990 Lys Asn Val Asp Phe Ser Lys Leu Ser Pro GluAla Leu Glu Ala Ile 995 1000 1005 Gly Lys Ser Ala Leu Glu Tyr Asp GlnArg Glu Asn Ala Thr Val Asp 1010 1015 1020 Asp Ile Ala Asn Ala Ala SerLeu Ile Glu Arg Asn Leu Arg Asn Ile 1025 1030 1035 1040 Ala Leu Gly ValAsp Ile Arg His Lys Val Leu Asp Lys Val Asn Leu 1045 1050 1055 Ser IleAsp Gln Met Ala Ala Val Gly Ala Pro Tyr Gln Asn Asn Gly 1060 1065 1070Lys Ile Asp Leu Ser Asn Met Thr Pro Glu Gln Gln Ala Asp Glu Leu 10751080 1085 Asn Lys Leu Phe Arg Glu Glu Leu Glu Ala Arg Lys Gln Lys ValAla 1090 1095 1100 Lys Ala Arg 1105 5 3432 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 5 atggggggttctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60 atgggtcgggatctgtacga cgatgacgat aaggatccga gctcgagatc tgaaagtaca 120 gttacagaagaattaaaaga aggtattgat gctgtttacc cttcattggt aggtactgct 180 gattctaaagcagagggtat taagaactat ttcaaattgt cctttacctt accagaagaa 240 cagaaatcccgtactgttgg ttcagaagca cctctaaaag atgtagccca agctctgtct 300 tctcgtgctcgttatgaact ctttactgag aaagaaactg ctaaccctgc ttttaatggg 360 gaagttattaagcgatacaa agaactcatg gaacatgggg aaggtattgc tgatattctt 420 cgctcccgtctggctaagtt ccttaacact aaggatgttg gtaaacgttt tgctcaaggt 480 acagaagccaaccgttgggt aggtggtaag ttacttaaca ttgttgagca ggatggggat 540 acctttaagtacaacgaaca attgctacag actgctgtat tagcaggtct tcaatggaga 600 cttactgctaccagcaatac tgctatcaaa gatgcaaaag atgttgctgc tattactggt 660 attgaccaagctctgctgcc agaaggttta gtagagcaat ttgatactgg tatgacactc 720 actgaagcagttagttccct ggctcagaaa attgagtctt actggggatt atctcgtaat 780 ccaaatgctccattgggcta taccaaaggc atccctacag caatggctgc tgaaattctg 840 gctgcatttgtagagtctac tgatgttgta gagaacatcg tggatatgtc agaaattgac 900 ccagataacaagaagactat tggtctgtac accattactg aactggattc cttcgaccca 960 attaatagcttccctactgc tattgaagaa gctgttttag tgaatcctac agagaagatg 1020 ttctttggtgatgacattcc tcctgtagct aatactcagc ttcgtaaccc tgctgttcgt 1080 aatactccagaacagaaggc tgcattgaaa gcagagcagg ctacagagtt ctatgtacac 1140 accccaatggttcaattcta tgagacgtta ggtaaagacc gtattctcga actgatgggt 1200 gctggtactctgaataaaga gttacttaat gataaccatg ctaaatctct ggaaggtaag 1260 aaccgttcagtagaggactc ttacaaccaa ctgttctccg tcattgagca ggtaagagca 1320 cagagcgaagacatctctac tgtacctatt cactatgcat acaatatgac ccgtgttggt 1380 cgtatgcagatgttaggtaa atacaatcct caatcagcca aactggttcg tgaggccatc 1440 ttacctactaaagctacttt ggatttatcg aaccagaaca atgaagactt ctctgcattc 1500 cagttaggtctggctcaggc attggacatt aaagtccata ctatgactcg tgaggttatg 1560 tctgacgagttgactaaatt actggaaggt aatctgaaac cagccattga tatgatggtt 1620 gagtttaataccactggttc cttaccagaa aacgcagttg atgttctgaa tacagcatta 1680 ggagataggaagtcattcgt agcattgatg gctcttatgg agtattcccg ttacttagta 1740 gcagaggataaatctgcatt tgtaactcca ctgtatgtag aagcagatgg tgttactaat 1800 ggtccaatcaatgccatgat gctaatgaca ggcggtctgt ttactcctga ctggattcgt 1860 aatattgccaaagggggctt gttcattggt tctccaaata agaccatgaa tgagcatcgc 1920 tctactgctgacaataatga tttatatcaa gcatccacta atgctttgat ggaatcgttg 1980 ggtaagttacgtagtaacta tgcctctaat atgcctattc agtctcagat agacagtctt 2040 ctttctctgatggatttgtt tttaccggat attaatcttg gtgagaatgg tgctttagaa 2100 cttaaacgtggtattgctaa gaacccactg actattacca tctatggttc tggtgctcgt 2160 ggtattgcaggtaagctggt tagttctgtt actgatgcca tctatgagcg tatgtctgat 2220 gtactgaaagctcgtgctaa agacccaaat atctctgctg ctatggcaat gtttggtaag 2280 caagctgcttcagaagcaca tgctgaagaa cttcttgccc gtttcctgaa agatatggaa 2340 acactgacttctactgttcc tgttaaacgt aaaggtgtac tggaactaca atccacaggt 2400 acaggagccaaaggaaaaat caatcctaag acctatacca ttaagggcga gcaactgaag 2460 gcacttcaggaaaatatgct gcacttcttt gtagaaccac tacgtaatgg tattactcag 2520 actgtaggtgaaagtctggt gtactctact gaacaattac agaaagctac tcagattcaa 2580 tctgtagtgctggaagatat gttcaaacag cgagtacaag agaagctggc agagaaggct 2640 aaagacccaacatggaagaa aggtgatttc cttactcaga aagaactgaa tgatattcag 2700 gcttctctgaataacttagc ccctatgatt gagactggtt ctcagacttt ctacattgct 2760 ggttcagaaaatgcagaagt agcaaatcag gtattagcta ctaaccttga tgaccgtatg 2820 cgtgtaccaatgagtatcta tgctccagca caggccggtg tagcaggtat tccatttatg 2880 actattggtactggtgatgg catgatgatg caaactcttt ccactatgaa aggtgcacca 2940 aagaataccctcaaaatctt tgatggtatg aacattggtt tgaatgacat cactgatgcc 3000 agtcgtaaagctaatgaagc tgtttacact tcttggcagg gtaaccctat taagaatgtt 3060 tatgaatcatatgctaagtt catgaagaat gtagatttca gcaagctgtc ccctgaagca 3120 ttggaagcaattggtaaatc tgctctggaa tatgaccaac gtgagaatgc tactgtagat 3180 gatattgctaacgctgcatc tctgattgaa cgtaacttac gtaatattgc actgggtgta 3240 gatattcgtcataaggtgct ggataaggta aatctgtcca ttgaccagat ggctgctgta 3300 ggtgctccttatcagaacaa cggtaagatt gacctcagca atatgacccc tgaacaacag 3360 gctgatgaactgaataaact tttccgtgaa gagttagaag cccgtaaaca aaaagtcgct 3420 aaggctaggtaa 3432 6 1143 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 6 Met Gly Gly Ser His His His His His His GlyMet Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu TyrAsp Asp Asp Asp Lys Asp 20 25 30 Pro Ser Ser Arg Ser Glu Ser Thr Val ThrGlu Glu Leu Lys Glu Gly 35 40 45 Ile Asp Ala Val Tyr Pro Ser Leu Val GlyThr Ala Asp Ser Lys Ala 50 55 60 Glu Gly Ile Lys Asn Tyr Phe Lys Leu SerPhe Thr Leu Pro Glu Glu 65 70 75 80 Gln Lys Ser Arg Thr Val Gly Ser GluAla Pro Leu Lys Asp Val Ala 85 90 95 Gln Ala Leu Ser Ser Arg Ala Arg TyrGlu Leu Phe Thr Glu Lys Glu 100 105 110 Thr Ala Asn Pro Ala Phe Asn GlyGlu Val Ile Lys Arg Tyr Lys Glu 115 120 125 Leu Met Glu His Gly Glu GlyIle Ala Asp Ile Leu Arg Ser Arg Leu 130 135 140 Ala Lys Phe Leu Asn ThrLys Asp Val Gly Lys Arg Phe Ala Gln Gly 145 150 155 160 Thr Glu Ala AsnArg Trp Val Gly Gly Lys Leu Leu Asn Ile Val Glu 165 170 175 Gln Asp GlyAsp Thr Phe Lys Tyr Asn Glu Gln Leu Leu Gln Thr Ala 180 185 190 Val LeuAla Gly Leu Gln Trp Arg Leu Thr Ala Thr Ser Asn Thr Ala 195 200 205 IleLys Asp Ala Lys Asp Val Ala Ala Ile Thr Gly Ile Asp Gln Ala 210 215 220Leu Leu Pro Glu Gly Leu Val Glu Gln Phe Asp Thr Gly Met Thr Leu 225 230235 240 Thr Glu Ala Val Ser Ser Leu Ala Gln Lys Ile Glu Ser Tyr Trp Gly245 250 255 Leu Ser Arg Asn Pro Asn Ala Pro Leu Gly Tyr Thr Lys Gly IlePro 260 265 270 Thr Ala Met Ala Ala Glu Ile Leu Ala Ala Phe Val Glu SerThr Asp 275 280 285 Val Val Glu Asn Ile Val Asp Met Ser Glu Ile Asp ProAsp Asn Lys 290 295 300 Lys Thr Ile Gly Leu Tyr Thr Ile Thr Glu Leu AspSer Phe Asp Pro 305 310 315 320 Ile Asn Ser Phe Pro Thr Ala Ile Glu GluAla Val Leu Val Asn Pro 325 330 335 Thr Glu Lys Met Phe Phe Gly Asp AspIle Pro Pro Val Ala Asn Thr 340 345 350 Gln Leu Arg Asn Pro Ala Val ArgAsn Thr Pro Glu Gln Lys Ala Ala 355 360 365 Leu Lys Ala Glu Gln Ala ThrGlu Phe Tyr Val His Thr Pro Met Val 370 375 380 Gln Phe Tyr Glu Thr LeuGly Lys Asp Arg Ile Leu Glu Leu Met Gly 385 390 395 400 Ala Gly Thr LeuAsn Lys Glu Leu Leu Asn Asp Asn His Ala Lys Ser 405 410 415 Leu Glu GlyLys Asn Arg Ser Val Glu Asp Ser Tyr Asn Gln Leu Phe 420 425 430 Ser ValIle Glu Gln Val Arg Ala Gln Ser Glu Asp Ile Ser Thr Val 435 440 445 ProIle His Tyr Ala Tyr Asn Met Thr Arg Val Gly Arg Met Gln Met 450 455 460Leu Gly Lys Tyr Asn Pro Gln Ser Ala Lys Leu Val Arg Glu Ala Ile 465 470475 480 Leu Pro Thr Lys Ala Thr Leu Asp Leu Ser Asn Gln Asn Asn Glu Asp485 490 495 Phe Ser Ala Phe Gln Leu Gly Leu Ala Gln Ala Leu Asp Ile LysVal 500 505 510 His Thr Met Thr Arg Glu Val Met Ser Asp Glu Leu Thr LysLeu Leu 515 520 525 Glu Gly Asn Leu Lys Pro Ala Ile Asp Met Met Val GluPhe Asn Thr 530 535 540 Thr Gly Ser Leu Pro Glu Asn Ala Val Asp Val LeuAsn Thr Ala Leu 545 550 555 560 Gly Asp Arg Lys Ser Phe Val Ala Leu MetAla Leu Met Glu Tyr Ser 565 570 575 Arg Tyr Leu Val Ala Glu Asp Lys SerAla Phe Val Thr Pro Leu Tyr 580 585 590 Val Glu Ala Asp Gly Val Thr AsnGly Pro Ile Asn Ala Met Met Leu 595 600 605 Met Thr Gly Gly Leu Phe ThrPro Asp Trp Ile Arg Asn Ile Ala Lys 610 615 620 Gly Gly Leu Phe Ile GlySer Pro Asn Lys Thr Met Asn Glu His Arg 625 630 635 640 Ser Thr Ala AspAsn Asn Asp Leu Tyr Gln Ala Ser Thr Asn Ala Leu 645 650 655 Met Glu SerLeu Gly Lys Leu Arg Ser Asn Tyr Ala Ser Asn Met Pro 660 665 670 Ile GlnSer Gln Ile Asp Ser Leu Leu Ser Leu Met Asp Leu Phe Leu 675 680 685 ProAsp Ile Asn Leu Gly Glu Asn Gly Ala Leu Glu Leu Lys Arg Gly 690 695 700Ile Ala Lys Asn Pro Leu Thr Ile Thr Ile Tyr Gly Ser Gly Ala Arg 705 710715 720 Gly Ile Ala Gly Lys Leu Val Ser Ser Val Thr Asp Ala Ile Tyr Glu725 730 735 Arg Met Ser Asp Val Leu Lys Ala Arg Ala Lys Asp Pro Asn IleSer 740 745 750 Ala Ala Met Ala Met Phe Gly Lys Gln Ala Ala Ser Glu AlaHis Ala 755 760 765 Glu Glu Leu Leu Ala Arg Phe Leu Lys Asp Met Glu ThrLeu Thr Ser 770 775 780 Thr Val Pro Val Lys Arg Lys Gly Val Leu Glu LeuGln Ser Thr Gly 785 790 795 800 Thr Gly Ala Lys Gly Lys Ile Asn Pro LysThr Tyr Thr Ile Lys Gly 805 810 815 Glu Gln Leu Lys Ala Leu Gln Glu AsnMet Leu His Phe Phe Val Glu 820 825 830 Pro Leu Arg Asn Gly Ile Thr GlnThr Val Gly Glu Ser Leu Val Tyr 835 840 845 Ser Thr Glu Gln Leu Gln LysAla Thr Gln Ile Gln Ser Val Val Leu 850 855 860 Glu Asp Met Phe Lys GlnArg Val Gln Glu Lys Leu Ala Glu Lys Ala 865 870 875 880 Lys Asp Pro ThrTrp Lys Lys Gly Asp Phe Leu Thr Gln Lys Glu Leu 885 890 895 Asn Asp IleGln Ala Ser Leu Asn Asn Leu Ala Pro Met Ile Glu Thr 900 905 910 Gly SerGln Thr Phe Tyr Ile Ala Gly Ser Glu Asn Ala Glu Val Ala 915 920 925 AsnGln Val Leu Ala Thr Asn Leu Asp Asp Arg Met Arg Val Pro Met 930 935 940Ser Ile Tyr Ala Pro Ala Gln Ala Gly Val Ala Gly Ile Pro Phe Met 945 950955 960 Thr Ile Gly Thr Gly Asp Gly Met Met Met Gln Thr Leu Ser Thr Met965 970 975 Lys Gly Ala Pro Lys Asn Thr Leu Lys Ile Phe Asp Gly Met AsnIle 980 985 990 Gly Leu Asn Asp Ile Thr Asp Ala Ser Arg Lys Ala Asn GluAla Val 995 1000 1005 Tyr Thr Ser Trp Gln Gly Asn Pro Ile Lys Asn ValTyr Glu Ser Tyr 1010 1015 1020 Ala Lys Phe Met Lys Asn Val Asp Phe SerLys Leu Ser Pro Glu Ala 1025 1030 1035 1040 Leu Glu Ala Ile Gly Lys SerAla Leu Glu Tyr Asp Gln Arg Glu Asn 1045 1050 1055 Ala Thr Val Asp AspIle Ala Asn Ala Ala Ser Leu Ile Glu Arg Asn 1060 1065 1070 Leu Arg AsnIle Ala Leu Gly Val Asp Ile Arg His Lys Val Leu Asp 1075 1080 1085 LysVal Asn Leu Ser Ile Asp Gln Met Ala Ala Val Gly Ala Pro Tyr 1090 10951100 Gln Asn Asn Gly Lys Ile Asp Leu Ser Asn Met Thr Pro Glu Gln Gln1105 1110 1115 1120 Ala Asp Glu Leu Asn Lys Leu Phe Arg Glu Glu Leu GluAla Arg Lys 1125 1130 1135 Gln Lys Val Ala Lys Ala Arg 1140 7 3432 DNAArtificial Sequence Description of Artificial Sequence Synthetic Primer7 atggggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60atgggtcggg atctgtacga cgatgacgat aaggatccga gctcgagatc tgaaagtaca 120gttacagaag aattaaaaga aggtattgat gctgtttacc cttcattggt aggtactgct 180gattctaaag cagagggtat taagaactat ttcaaattgt cctttacctt accagaagaa 240cagaaatccc gtactgttgg ttcagaagca cctctaaaag atgtagccca agctctgtct 300tctcgtgctc gttatgaact ctttactgag aaagaaactg ctaaccctgc ttttaatggg 360gaagttatta agcgatacaa agaactcatg gaacatgggg aaggtattgc tgatattctt 420cgctcccgtc tggctaagtt ccttaacact aaggatgttg gtaaacgttt tgctcaaggt 480acagaagcca accgttgggt aggtggtaag ttacttaaca ttgttgagca ggatggggat 540acctttaagt acaacgaaca attgctacag actgctgtat tagcaggtct tcaatggaga 600cttactgcta ccagcaatac tgctatcaaa gatgcaaaag atgttgctgc tattactggt 660attgaccaag ctctgctgcc agaaggttta gtagagcaat ttgatactgg tatgacactc 720actgaagcag ttagttccct ggctcagaaa attgagtctt actggggatt atctcgtaat 780ccaaatgctc cattgggcta taccaaaggc atccctacag caatggctgc tgaaattctg 840gctgcatttg tagagtctac tgatgttgta gagaacatcg tggatatgtc agaaattgac 900ccagataaca agaagactat tggtctgtac accattactg aactggattc cttcgaccca 960attaatagct tccctactgc tattgaagaa gctgttttag tgaatcctac agagaagatg 1020ttctttggtg atgacattcc tcctgtagct aatactcagc ttcgtaaccc tgctgttcgt 1080aatactccag aacagaaggc tgcattgaaa gcagagcagg ctacagagtt ctatgtacac 1140accccaatgg ttcaattcta tgagacgtta ggtaaagacc gtattctcga actgatgggt 1200gctggtactc tgaataaaga gttacttaat gataaccatg ctaaatctct ggaaggtaag 1260aaccgttcag tagaggactc ttacaaccaa ctgttctccg tcattgagca ggtaagagca 1320cagagcgaag acatctctac tgtacctatt cactatgcat acaatatgac ccgtgttggt 1380cgtatgcaga tgttaggtaa atacaatcct caatcagcca aactggttcg tgaggccatc 1440ttacctacta aagctacttt ggatttatcg aaccagaaca atgaagactt ctctgcattc 1500cagttaggtc tggctcaggc attggacatt aaagtccata ctatgactcg tgaggttatg 1560tctgacgagt tgactaaatt actggaaggt aatctgaaac cagccattga tatgatggtt 1620gagtttaata ccactggttc cttaccagaa aacgcagttg atgttctgaa tacagcatta 1680ggagatagga agtcattcgt agcattgatg gctcttatgg agtattcccg ttacttagta 1740gcagaggata aatctgcatt tgtaactcca ctgtatgtag aagcagatgg tgttactaat 1800ggtccaatca atgccatgat gctaatgaca ggcggtctgt ttactcctga ctggattcgt 1860aatattgcca aagggggctt gttcattggt tctccaaata agaccatgaa tgagcatcgc 1920tctactgctg acaataatga tttatatcaa gcatccacta atgctttgat ggaatcgttg 1980ggtaagttac gtagtaacta tgcctctaat atgcctattc agtctcagat agacagtctt 2040ctttctctga tggatttgtt tttaccggat attaatcttg gtgagaatgg tgctttagaa 2100cttaaacgtg gtattgctaa gaacccactg actattacca tcttcggttc tggtgctcgt 2160ggtattgcag gtaagctggt tagttctgtt actgatgcca tctatgagcg tatgtctgat 2220gtactgaaag ctcgtgctaa agacccaaat atctctgctg ctatggcaat gtttggtaag 2280caagctgctt cagaagcaca tgctgaagaa cttcttgccc gtttcctgaa agatatggaa 2340acactgactt ctactgttcc tgttaaacgt aaaggtgtac tggaactaca atccacaggt 2400acaggagcca aaggaaaaat caatcctaag acctatacca ttaagggcga gcaactgaag 2460gcacttcagg aaaatatgct gcacttcttt gtagaaccac tacgtaatgg tattactcag 2520actgtaggtg aaagtctggt gtactctact gaacaattac agaaagctac tcagattcaa 2580tctgtagtgc tggaagatat gttcaaacag cgagtacaag agaagctggc agagaaggct 2640aaagacccaa catggaagaa aggtgatttc cttactcaga aagaactgaa tgatattcag 2700gcttctctga ataacttagc ccctatgatt gagactggtt ctcagacttt ctacattgct 2760ggttcagaaa atgcagaagt agcaaatcag gtattagcta ctaaccttga tgaccgtatg 2820cgtgtaccaa tgagtatcta tgctccagca caggccggtg tagcaggtat tccatttatg 2880actattggta ctggtgatgg catgatgatg caaactcttt ccactatgaa aggtgcacca 2940aagaataccc tcaaaatctt tgatggtatg aacattggtt tgaatgacat cactgatgcc 3000agtcgtaaag ctaatgaagc tgtttacact tcttggcagg gtaaccctat taagaatgtt 3060tatgaatcat atgctaagtt catgaagaat gtagatttca gcaagctgtc ccctgaagca 3120ttggaagcaa ttggtaaatc tgctctggaa tatgaccaac gtgagaatgc tactgtagat 3180gatattgcta acgctgcatc tctgattgaa cgtaacttac gtaatattgc actgggtgta 3240gatattcgtc ataaggtgct ggataaggta aatctgtcca ttgaccagat ggctgctgta 3300ggtgctcctt atcagaacaa cggtaagatt gacctcagca atatgacccc tgaacaacag 3360gctgatgaac tgaataaact tttccgtgaa gagttagaag cccgtaaaca aaaagtcgct 3420aaggctaggt aa 3432 8 1143 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 8 Met Gly Gly Ser His His His HisHis His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly ArgAsp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Ser Ser Arg Ser Glu SerThr Val Thr Glu Glu Leu Lys Glu Gly 35 40 45 Ile Asp Ala Val Tyr Pro SerLeu Val Gly Thr Ala Asp Ser Lys Ala 50 55 60 Glu Gly Ile Lys Asn Tyr PheLys Leu Ser Phe Thr Leu Pro Glu Glu 65 70 75 80 Gln Lys Ser Arg Thr ValGly Ser Glu Ala Pro Leu Lys Asp Val Ala 85 90 95 Gln Ala Leu Ser Ser ArgAla Arg Tyr Glu Leu Phe Thr Glu Lys Glu 100 105 110 Thr Ala Asn Pro AlaPhe Asn Gly Glu Val Ile Lys Arg Tyr Lys Glu 115 120 125 Leu Met Glu HisGly Glu Gly Ile Ala Asp Ile Leu Arg Ser Arg Leu 130 135 140 Ala Lys PheLeu Asn Thr Lys Asp Val Gly Lys Arg Phe Ala Gln Gly 145 150 155 160 ThrGlu Ala Asn Arg Trp Val Gly Gly Lys Leu Leu Asn Ile Val Glu 165 170 175Gln Asp Gly Asp Thr Phe Lys Tyr Asn Glu Gln Leu Leu Gln Thr Ala 180 185190 Val Leu Ala Gly Leu Gln Trp Arg Leu Thr Ala Thr Ser Asn Thr Ala 195200 205 Ile Lys Asp Ala Lys Asp Val Ala Ala Ile Thr Gly Ile Asp Gln Ala210 215 220 Leu Leu Pro Glu Gly Leu Val Glu Gln Phe Asp Thr Gly Met ThrLeu 225 230 235 240 Thr Glu Ala Val Ser Ser Leu Ala Gln Lys Ile Glu SerTyr Trp Gly 245 250 255 Leu Ser Arg Asn Pro Asn Ala Pro Leu Gly Tyr ThrLys Gly Ile Pro 260 265 270 Thr Ala Met Ala Ala Glu Ile Leu Ala Ala PheVal Glu Ser Thr Asp 275 280 285 Val Val Glu Asn Ile Val Asp Met Ser GluIle Asp Pro Asp Asn Lys 290 295 300 Lys Thr Ile Gly Leu Tyr Thr Ile ThrGlu Leu Asp Ser Phe Asp Pro 305 310 315 320 Ile Asn Ser Phe Pro Thr AlaIle Glu Glu Ala Val Leu Val Asn Pro 325 330 335 Thr Glu Lys Met Phe PheGly Asp Asp Ile Pro Pro Val Ala Asn Thr 340 345 350 Gln Leu Arg Asn ProAla Val Arg Asn Thr Pro Glu Gln Lys Ala Ala 355 360 365 Leu Lys Ala GluGln Ala Thr Glu Phe Tyr Val His Thr Pro Met Val 370 375 380 Gln Phe TyrGlu Thr Leu Gly Lys Asp Arg Ile Leu Glu Leu Met Gly 385 390 395 400 AlaGly Thr Leu Asn Lys Glu Leu Leu Asn Asp Asn His Ala Lys Ser 405 410 415Leu Glu Gly Lys Asn Arg Ser Val Glu Asp Ser Tyr Asn Gln Leu Phe 420 425430 Ser Val Ile Glu Gln Val Arg Ala Gln Ser Glu Asp Ile Ser Thr Val 435440 445 Pro Ile His Tyr Ala Tyr Asn Met Thr Arg Val Gly Arg Met Gln Met450 455 460 Leu Gly Lys Tyr Asn Pro Gln Ser Ala Lys Leu Val Arg Glu AlaIle 465 470 475 480 Leu Pro Thr Lys Ala Thr Leu Asp Leu Ser Asn Gln AsnAsn Glu Asp 485 490 495 Phe Ser Ala Phe Gln Leu Gly Leu Ala Gln Ala LeuAsp Ile Lys Val 500 505 510 His Thr Met Thr Arg Glu Val Met Ser Asp GluLeu Thr Lys Leu Leu 515 520 525 Glu Gly Asn Leu Lys Pro Ala Ile Asp MetMet Val Glu Phe Asn Thr 530 535 540 Thr Gly Ser Leu Pro Glu Asn Ala ValAsp Val Leu Asn Thr Ala Leu 545 550 555 560 Gly Asp Arg Lys Ser Phe ValAla Leu Met Ala Leu Met Glu Tyr Ser 565 570 575 Arg Tyr Leu Val Ala GluAsp Lys Ser Ala Phe Val Thr Pro Leu Tyr 580 585 590 Val Glu Ala Asp GlyVal Thr Asn Gly Pro Ile Asn Ala Met Met Leu 595 600 605 Met Thr Gly GlyLeu Phe Thr Pro Asp Trp Ile Arg Asn Ile Ala Lys 610 615 620 Gly Gly LeuPhe Ile Gly Ser Pro Asn Lys Thr Met Asn Glu His Arg 625 630 635 640 SerThr Ala Asp Asn Asn Asp Leu Tyr Gln Ala Ser Thr Asn Ala Leu 645 650 655Met Glu Ser Leu Gly Lys Leu Arg Ser Asn Tyr Ala Ser Asn Met Pro 660 665670 Ile Gln Ser Gln Ile Asp Ser Leu Leu Ser Leu Met Asp Leu Phe Leu 675680 685 Pro Asp Ile Asn Leu Gly Glu Asn Gly Ala Leu Glu Leu Lys Arg Gly690 695 700 Ile Ala Lys Asn Pro Leu Thr Ile Thr Ile Phe Gly Ser Gly AlaArg 705 710 715 720 Gly Ile Ala Gly Lys Leu Val Ser Ser Val Thr Asp AlaIle Tyr Glu 725 730 735 Arg Met Ser Asp Val Leu Lys Ala Arg Ala Lys AspPro Asn Ile Ser 740 745 750 Ala Ala Met Ala Met Phe Gly Lys Gln Ala AlaSer Glu Ala His Ala 755 760 765 Glu Glu Leu Leu Ala Arg Phe Leu Lys AspMet Glu Thr Leu Thr Ser 770 775 780 Thr Val Pro Val Lys Arg Lys Gly ValLeu Glu Leu Gln Ser Thr Gly 785 790 795 800 Thr Gly Ala Lys Gly Lys IleAsn Pro Lys Thr Tyr Thr Ile Lys Gly 805 810 815 Glu Gln Leu Lys Ala LeuGln Glu Asn Met Leu His Phe Phe Val Glu 820 825 830 Pro Leu Arg Asn GlyIle Thr Gln Thr Val Gly Glu Ser Leu Val Tyr 835 840 845 Ser Thr Glu GlnLeu Gln Lys Ala Thr Gln Ile Gln Ser Val Val Leu 850 855 860 Glu Asp MetPhe Lys Gln Arg Val Gln Glu Lys Leu Ala Glu Lys Ala 865 870 875 880 LysAsp Pro Thr Trp Lys Lys Gly Asp Phe Leu Thr Gln Lys Glu Leu 885 890 895Asn Asp Ile Gln Ala Ser Leu Asn Asn Leu Ala Pro Met Ile Glu Thr 900 905910 Gly Ser Gln Thr Phe Tyr Ile Ala Gly Ser Glu Asn Ala Glu Val Ala 915920 925 Asn Gln Val Leu Ala Thr Asn Leu Asp Asp Arg Met Arg Val Pro Met930 935 940 Ser Ile Tyr Ala Pro Ala Gln Ala Gly Val Ala Gly Ile Pro PheMet 945 950 955 960 Thr Ile Gly Thr Gly Asp Gly Met Met Met Gln Thr LeuSer Thr Met 965 970 975 Lys Gly Ala Pro Lys Asn Thr Leu Lys Ile Phe AspGly Met Asn Ile 980 985 990 Gly Leu Asn Asp Ile Thr Asp Ala Ser Arg LysAla Asn Glu Ala Val 995 1000 1005 Tyr Thr Ser Trp Gln Gly Asn Pro IleLys Asn Val Tyr Glu Ser Tyr 1010 1015 1020 Ala Lys Phe Met Lys Asn ValAsp Phe Ser Lys Leu Ser Pro Glu Ala 1025 1030 1035 1040 Leu Glu Ala IleGly Lys Ser Ala Leu Glu Tyr Asp Gln Arg Glu Asn 1045 1050 1055 Ala ThrVal Asp Asp Ile Ala Asn Ala Ala Ser Leu Ile Glu Arg Asn 1060 1065 1070Leu Arg Asn Ile Ala Leu Gly Val Asp Ile Arg His Lys Val Leu Asp 10751080 1085 Lys Val Asn Leu Ser Ile Asp Gln Met Ala Ala Val Gly Ala ProTyr 1090 1095 1100 Gln Asn Asn Gly Lys Ile Asp Leu Ser Asn Met Thr ProGlu Gln Gln 1105 1110 1115 1120 Ala Asp Glu Leu Asn Lys Leu Phe Arg GluGlu Leu Glu Ala Arg Lys 1125 1130 1135 Gln Lys Val Ala Lys Ala Arg 11409 69 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 9 tcccagacaa aaggttaaga tttcatacag gattggatgcattacttcat ccaaaagaag 60 cggagcttc 69 10 69 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 10 tgggagagaaaaggttaaga tttgatagag gattggatgg attagttgat ggaaaagaag 60 cggagcttc 6911 69 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 11 tccctgtctt ttggttttgt tttctttctg gtttggttgcttttcttctt ccaaaagaag 60 cggagcttc 69 12 69 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 12 tcccacacaaaaccttaaca tttcatacac cattccatcc attacttcat ccaaaagaag 60 cggagcttc 6913 69 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 13 acccagacaa aaggaaaaga aaacaaacag gaaaggaagcaaaacaacaa ccaaaagaag 60 cggagcttc 69 14 10617 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 14 atggggggttctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60 atgggtcgggatctgtacga cgatgacgat aaggatccga gctcgagatc tatgtcagta 120 tttgatagactggctgggtt cgcagacagc gtaaccaatg caaagcaagt tgacgtctct 180 actgcaaccgcccagaagaa agctgaacaa ggtgtcacta ctcctcttgt ttctcctgat 240 gctgcttatcaaatgcaagc tgcccgtact ggtaatgttg gggctaatgc atttgaacca 300 gggacagtgcaatcagattt catgaatctg accccaatgc aaatcatgaa taagtatggg 360 gttgagcaaggcttacaact tatcaatgct cgtgctgatg cagggaacca ggtattcaat 420 gattcagttactacaagaac tcctggggaa gaactggggg atattgctac tggtgttggc 480 cttggttttgttaataccct tgggggcatt ggtgctcttg gggcaggctt actcaacgat 540 gatgcaggtgctgttgttgc tcaacaattg agtaagttta atgatgctgt tcatgctacc 600 caaagccaggcattacaaga taaacgtaag ctctttgctg ctcgtaactt aatgaatgaa 660 gtagagagtgaacgtcagta tcaaacagat aagaaagaag gcactaatga catagtagct 720 tccttatctaaatttggacg tgattttgta ggttcaattg agaatgctgc tcaaactgac 780 tctattatttctgatgggtt agcagaaggg gtaggttctc tattaggtgc tggtcctgta 840 ttaaggggtgcatctttact gggtaaagca gttgttccag caaatactct tcgtagtgct 900 gcattggctggtgctattga tgcaggtact ggtactcagt cactggctcg tattgcctct 960 actgtaggtagagctgcacc gggtatggtt ggtgttggtg caatggaagc tggtggtgca 1020 taccaacaaactgctgatga aattatgaag atgagtctta aagacttaga gaagtctcct 1080 gtttatcagcaacatattaa agatggtatg tcccctgaac aggctcgtcg tcagactgca 1140 tctgaaactggtcttactgc tgctgctatt caattaccta ttgctgctgc aaccggtcct 1200 ctggtatcccgttttgagat ggctcctttc cgtgctggct ctttaggtgc tgtaggtatg 1260 aaccttgcccgtgaaacagt ggaagaaggt gttcagggtg ctacaggcca actggctcag 1320 aatattgcacagcaacaaaa cattgataag aaccaagacc tgcttaaagg tgtcggtaca 1380 caggctggtttaggtgctct ttatggcttt ggttctgctg gtgttgtaca ggctccggct 1440 ggtgctgctcgtttagcagg tgctgcaact gctcctgtat tgcgtaccac aatggctggt 1500 gttaaagctgctggtagtgt agcaggtaag gttgtttctc ctattaagaa tactttagta 1560 gctcgtggtgaacgggttat gaagcagaat gaagaagcat ctcctgttgc tgatgactat 1620 gttgcacaggcagcacaaga agctatggct caagcaccag aagcagaagt tactattcgt 1680 gatgctgttgaagcaactga tgctactcca gaacagaaag ttgcagcaca ccagtatgtt 1740 tctgacttaatgaatgctac tcgttttaat cctgaaaatt atcaggaagc accagagcat 1800 attcgtaatgctgtagctgg ttctactgac caagtacagg ttattcagaa gttagcagac 1860 ttagttaacacattagatga atctaatcct caagcactga tggaagctgc atcttatatg 1920 tatgatgctgtttcagagtt tgagcagttc attaaccgtg accctgctgc actggatagc 1980 attcctaaagattctccggc tattgagtta ctcaaccgtt atacgaatct gacagctaat 2040 attcagaacacaccaaaagt aattggtgca ctgaatgtta ttaatcgaat gattaatgaa 2100 tctgctcagaatggttcttt gaatgtgact gaagaatcca gtccacagga aatgcagaac 2160 gtagcattagctgctgaagt agcccctgaa aagctcaatc cagagtctgt aaatgttgtt 2220 cttaaacatgctgctgatgg tcgtattaaa ctgaataatc gccagattgc tgccctccag 2280 aatgctgctgcaatcctgaa gggggcacgg gaatatgatg cagaagctgc ccgtcttgga 2340 ttacgtcctcaagacattgt gagtaaacag attaaaacgg atgagagcag aactcaggaa 2400 ggacaatactctgcgttgca acatgcgaat aggattcggt ctgcgtataa ctctggtaat 2460 ttcgagttggcctccgctta cctgaacgac tttatgcagt tcgcccagca catgcagaat 2520 aaggttggagcgttgaatga gcatcttgtt acggggaatg cggataagaa taagtctgtc 2580 cactaccaagctcttactgc tgacagagaa tgggttcgta gccgtaccgg attgggggtc 2640 aatccctatgacactaagtc ggttaaattt gcccagcaag ttgctcttga agcgaaaacg 2700 gtagcggatattgctaatgc cctcgcttcg gcttacccgg aactgaaggt cagtcatata 2760 aaagttactccattggattc acgtcttaac gctcctgctg ctgaggtggt caaggcattc 2820 cgtcaaggcaatcgagacgt tgcttcttct caaccgaaag ctgactccgt gaatcaggtt 2880 aaagaaactcctgttacaaa acaggaacca gttacatcta ctgtacagac taagactcct 2940 gttagtgaatctgttaaaac agaacctact actaaagagt ctagcccaca ggctataaaa 3000 gaacctgtgaaccagtctga aaaacaggat gttaacctta ctaatgagga caacatcaag 3060 caacctactgaatctgttaa agaaactgaa acttctacaa aagaaagtac agttacagaa 3120 gaattaaaagaaggtattga tgctgtttac ccttcattgg taggtactgc tgattctaaa 3180 gcagagggtattaagaacta tttcaaattg tcctttacct taccagaaga acagaaatcc 3240 cgtactgttggttcagaagc acctctaaaa gatgtagccc aagctctgtc ttctcgtgct 3300 cgttatgaactctttactga gaaagaaact gctaaccctg cttttaatgg ggaagttatt 3360 aagcgatacaaagaactcat ggaacatggg gaaggtattg ctgatattct tcgctcccgt 3420 ctggctaagttccttaacac taaggatgtt ggtaaacgtt ttgctcaagg tacagaagcc 3480 aaccgttgggtaggtggtaa gttacttaac attgttgagc aggatgggga tacctttaag 3540 tacaacgaacaattgctaca gactgctgta ttagcaggtc ttcaatggag acttactgct 3600 accagcaatactgctatcaa agatgcaaaa gatgttgctg ctattactgg tattgaccaa 3660 gctctgctgccagaaggttt agtagagcaa tttgatactg gtatgacact cactgaagca 3720 gttagttccctggctcagaa aattgagtct tactggggat tatctcgtaa tccaaatgct 3780 ccattgggctataccaaagg catccctaca gcaatggctg ctgaaattct ggctgcattt 3840 gtagagtctactgatgttgt agagaacatc gtggatatgt cagaaattga cccagataac 3900 aagaagactattggtctgta caccattact gaactggatt ccttcgaccc aattaatagc 3960 ttccctactgctattgaaga agctgtttta gtgaatccta cagagaagat gttctttggt 4020 gatgacattcctcctgtagc taatactcag cttcgtaacc ctgctgttcg taatactcca 4080 gaacagaaggctgcattgaa agcagagcag gctacagagt tctatgtaca caccccaatg 4140 gttcaattctatgagacgtt aggtaaagac cgtattctcg aactgatggg tgctggtact 4200 ctgaataaagagttacttaa tgataaccat gctaaatctc tggaaggtaa gaaccgttca 4260 gtagaggactcttacaacca actgttctcc gtcattgagc aggtaagagc acagagcgaa 4320 gacatctctactgtacctat tcactatgca tacaatatga cccgtgttgg tcgtatgcag 4380 atgttaggtaaatacaatcc tcaatcagcc aaactggttc gtgaggccat cttacctact 4440 aaagctactttggatttatc gaaccagaac aatgaagact tctctgcatt ccagttaggt 4500 ctggctcaggcattggacat taaagtccat actatgactc gtgaggttat gtctgacgag 4560 ttgactaaattactggaagg taatctgaaa ccagccattg atatgatggt tgagtttaat 4620 accactggttccttaccaga aaacgcagtt gatgttctga atacagcatt aggagatagg 4680 aagtcattcgtagcattgat ggctcttatg gagtattccc gttacttagt agcagaggat 4740 aaatctgcatttgtaactcc actgtatgta gaagcagatg gtgttactaa tggtccaatc 4800 aatgccatgatgctaatgac aggcggtctg tttactcctg actggattcg taatattgcc 4860 aaagggggcttgttcattgg ttctccaaat aagaccatga atgagcatcg ctctactgct 4920 gacaataatgatttatatca agcatccact aatgctttga tggaatcgtt gggtaagtta 4980 cgtagtaactatgcctctaa tatgcctatt cagtctcaga tagacagtct tctttctctg 5040 atggatttgtttttaccgga tattaatctt ggtgagaatg gtgctttaga acttaaacgt 5100 ggtattgctaagaacccact gactattacc atctatggtt ctggtgctcg tggtattgca 5160 ggtaagctggttagttctgt tactgatgcc atctatgagc gtatgtctga tgtactgaaa 5220 gctcgtgctaaagacccaaa tatctctgct gctatggcaa tgtttggtaa gcaagctgct 5280 tcagaagcacatgctgaaga acttcttgcc cgtttcctga aagatatgga aacactgact 5340 tctactgttcctgttaaacg taaaggtgta ctggaactac aatccacagg tacaggagcc 5400 aaaggaaaaatcaatcctaa gacctatacc attaagggcg agcaactgaa ggcacttcag 5460 gaaaatatgctgcacttctt tgtagaacca ctacgtaatg gtattactca gactgtaggt 5520 gaaagtctggtgtactctac tgaacaatta cagaaagcta ctcagattca atctgtagtg 5580 ctggaagatatgttcaaaca gcgagtacaa gagaagctgg cagagaaggc taaagaccca 5640 acatggaagaaaggtgattt ccttactcag aaagaactga atgatattca ggcttctctg 5700 aataacttagcccctatgat tgagactggt tctcagactt tctacattgc tggttcagaa 5760 aatgcagaagtagcaaatca ggtattagct actaaccttg atgaccgtat gcgtgtacca 5820 atgagtatctatgctccagc acaggccggt gtagcaggta ttccatttat gactattggt 5880 actggtgatggcatgatgat gcaaactctt tccactatga aaggtgcacc aaagaatacc 5940 ctcaaaatctttgatggtat gaacattggt ttgaatgaca tcactgatgc cagtcgtaaa 6000 gctaatgaagctgtttacac ttcttggcag ggtaacccta ttaagaatgt ttatgaatca 6060 tatgctaagttcatgaagaa tgtagatttc agcaagctgt cccctgaagc attggaagca 6120 attggtaaatctgctctgga atatgaccaa cgtgagaatg ctactgtaga tgatattgct 6180 aacgctgcatctctgattga acgtaactta cgtaatattg cactgggtgt agatattcgt 6240 cataaggtgctggataaggt aaatctgtcc attgaccaga tggctgctgt aggtgctcct 6300 tatcagaacaacggtaagat tgacctcagc aatatgaccc ctgaacaaca ggctgatgaa 6360 ctgaataaacttttccgtga agagttagaa gcccgtaaac aaaaagtcgc taaggctagg 6420 gctgaagtcaaagaagaaac tgtttctgaa aaagaaccag tgaatccaga ctttggtatg 6480 gtaggccgtgagcataaggc atctggtgtt cgtatcctgt ctgctactgc tattcgtaat 6540 ctggctaagattagtaatct gccatctact caggcagcta ctcttgcgga gattcagaaa 6600 tcactggcagctaaagacta taagattatc tacggtacac ctactcaggt tgcagagtat 6660 gctcgtcagaagaatgttac tgaattgact tctcaggaaa tggaagaagc tcaggcaggt 6720 aatatttatggctggactaa cttcgatgat aagaccattt atctggttag cccatctatg 6780 gaaaccctcattcatgaact ggttcatgcc tctaccttcg aggaagttta ttccttctat 6840 cagggtaatgaagtaagccc tacttctaag caggctattg agaaccttga aggtctgatg 6900 gaacagttccgttctctgga tatttccaaa gattctccag aaatgagaga agcatatgct 6960 gatgctattgcaactatcga aggtcatttg agtaatggat ttgttgaccc agctatctct 7020 aaagctgctgctcttaatga gtttatggct tgggggttag ctaaccgtgc tcttgctgct 7080 aaacagaagagaacatcttc actggttcaa atggtgaaag atgtttatca ggctattaag 7140 aaattgatttggggacgtaa acaagctcct gcattgggag aagatatgtt ctccaatctg 7200 ctgtttaactctgcaattct gatgcgtagc caacctacaa ctcaggcagt agctaaagat 7260 ggcacactgttccatagcaa agcatatggt aataatgaac gtctgtctca gttgaaccag 7320 actttcgataaactggtaac tgattacctt cgtactgacc cagttacaga agtagaacgt 7380 cgtggcaatgtggctaatgc attaatgagt gctactcgac tggttcgtga tgttcagtct 7440 catggcttcaatatgactgc tcaggaacag tctgtattcc agatggttac tgctgcatta 7500 gcaactgaagctgcgattga cccacatgct atggctcgtg ctcaggaact ttatacccat 7560 gtaatgaaacaccttacggt agagcatttc atggctgacc ctgatagtac taaccctgct 7620 gaccgttactatgctcaaca gaaatatgac accatctctg gtgctaatct ggttgaagta 7680 gatgccaaaggtagaaccag tctgttacct acattcctgg gtctggctat ggttaatgaa 7740 gaactacgttcaatcattaa agaaatgcct gtacctaaag cagataagaa attagggaat 7800 gatatagatactctgcttac caatgcaggt actcaggtaa tggaatctct gaaccgtcgt 7860 atggctggtgaccagaaagc tactaatgtt caggacagta ttgatgcttt gtcagaaaca 7920 atcatggctgctgctttgaa acgagagtcc ttctatgatg ctgtagcaac ccctaccggt 7980 aacttcattgaccgtgctaa tcagtacgta acggatagca ttgaacggtt atctgaaact 8040 gttattgagaaggcagataa ggtaattgct aacccttcta atatagctgc taaaggtgtt 8100 gctcatctggctaaactgac tgctgctatt gcatctgaaa aacagggtga aatagtggct 8160 cagggtgttatgactgctat gaaccagggt aaagtatggc aacctttcca tgacttagtt 8220 aatgacattgttggccgtac taagactaat gccaatgtct atgacttaat caaattggtt 8280 aagagccagatttctcaaga ccgtcagcaa ttccgtgagc atttacctac agtcattgct 8340 ggtaagttctctcgtaaatt gactgatacc gaatggtctg caatgcatac tggtttaggt 8400 aaaacagatttagctgttct acgtgaaact atgagcatgg ctgaaattag agatttactc 8460 tcttcatccaagaaagtgaa agatgaaatc tctactctgg aaaaagagat tcagaaccaa 8520 gcaggtagaaactggaatct ggttcagaag aaatctaagc aactggctca atacatgatt 8580 atgggggaagtaggtaataa cctccttcgt aatgcccatg ctattagtcg tttgttaggt 8640 gaacgtattactaatggtcc tgtggcagat gtagctgcta ttgataagct cattactttg 8700 tactctctggaattgatgaa taagtctgac cgtgaccttt tgtcagaatt ggctcaatca 8760 gaagtggaaggtatggagtt ctccattgct tatatggttg gtcaacgtac tgaagagatg 8820 cgtaaagctaaaggtgataa ccgtactctg ctgaatcact ttaaaggcta tatccctgta 8880 gagaaccagcaaggtgtgaa tttgattatt gctgacgata aagagtttgc taagttaaat 8940 agccaatcctttactcgtat tggtacttat caggggagca ctggtttccg tactggttct 9000 aaaggttattacttcagccc agtagctgcc cgtgcccctt actctcaggg tattcttcag 9060 aacgttcgtaatactgctgg tggtgtggat attggtactg gctttacgtt aggcactatg 9120 gttgctgggcgtattactga caaaccaacc gtagagcgta ttaccaaagc tctggctaaa 9180 ggtgagcgtgggcgtgaacc actgatgcca atttataaca gcaaaggtca ggtagttgct 9240 tatgaacaatccgttgaccc taatatgttg aagcacctaa accaagacaa tcactttgct 9300 aagatggttggtgtatggcg tggtcgtcag gtggaagagg ctaaagcaca acgttttaat 9360 gacattctcattgagcaatt acatgctatg tatgagaaag acattaaaga ctccagtgct 9420 aataaatctcaatatgtaaa cctgttaggt aaaattgatg acccagtact ggctgatgcg 9480 attaacctgatgaacattga gactcgtcat aaggccgaag aactcttcgg taaagatgag 9540 ttatgggttcgtagggatat gctgaatgat gcacttggct atcgtgctgc atctattggt 9600 gatgtgtggaccggtaactc tcgttggtca cctagcaccc ttgatactgt taagaagatg 9660 ttcctcggtgcattcggtaa taaggcatat catgtagtaa tgaatgctga aaataccatt 9720 cagaacttagtgaaggacgc taagacagta attgttgtta aatctgttgt agtaccggca 9780 gttaacttccttgctaacat ctaccagatg attggacgtg gtgttcctgt taaagatatt 9840 gctgtgaacattcctcgtaa gacgtcagag attaatcagt atattaaatc tcgtttacgt 9900 cagattgatgcggaagcaga gctacgtgct gctgaaggta accctaatct ggttcgtaaa 9960 cttaaaactgagattcaatc tattactgat agtcatcgtc gtatgagtat ctggcctttg 10020 attgaagcaggtgagttctc ttctattgct gatgctggta ttagtcgtga tgacctgtta 10080 gtagctgaaggtaagattca tgagtacatg gaaaaacttg ctaataaact tccagaaaaa 10140 gtacgtaatgctggccgtta cgctcttatt gctaaggaca ctgctctgtt ccagggtatc 10200 cagaaaacagtagagtattc agactttatt gctaaagcca tcatctatga tgatttagtg 10260 aaacgtaagaaaaaatcttc ttctgaagca ttaggtcagg taactgaaga gtttattaac 10320 tatgacagattgcctggtcg tttccgtggc tatatggaaa gtatgggtct gatgtggttc 10380 tacaactttaaaattcgttc cattaaagtt gctatgagca tgattagaaa caacccagta 10440 cattctctgattgctacagt agtacctgct cctaccatgt ttggtaacgt aggtctacca 10500 attcaggacaacatgctaac catgctggct gaaggaagac tggattactc attaggcttc 10560 ggacaaggattaagagcacc taccctcaat ccttggttca accttactca ctaataa 10617 15 3537 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide15 Met Gly Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 510 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 2025 30 Pro Ser Ser Arg Ser Met Ser Val Phe Asp Arg Leu Ala Gly Phe Ala 3540 45 Asp Ser Val Thr Asn Ala Lys Gln Val Asp Val Ser Thr Ala Thr Ala 5055 60 Gln Lys Lys Ala Glu Gln Gly Val Thr Thr Pro Leu Val Ser Pro Asp 6570 75 80 Ala Ala Tyr Gln Met Gln Ala Ala Arg Thr Gly Asn Val Gly Ala Asn85 90 95 Ala Phe Glu Pro Gly Thr Val Gln Ser Asp Phe Met Asn Leu Thr Pro100 105 110 Met Gln Ile Met Asn Lys Tyr Gly Val Glu Gln Gly Leu Gln LeuIle 115 120 125 Asn Ala Arg Ala Asp Ala Gly Asn Gln Val Phe Asn Asp SerVal Thr 130 135 140 Thr Arg Thr Pro Gly Glu Glu Leu Gly Asp Ile Ala ThrGly Val Gly 145 150 155 160 Leu Gly Phe Val Asn Thr Leu Gly Gly Ile GlyAla Leu Gly Ala Gly 165 170 175 Leu Leu Asn Asp Asp Ala Gly Ala Val ValAla Gln Gln Leu Ser Lys 180 185 190 Phe Asn Asp Ala Val His Ala Thr GlnSer Gln Ala Leu Gln Asp Lys 195 200 205 Arg Lys Leu Phe Ala Ala Arg AsnLeu Met Asn Glu Val Glu Ser Glu 210 215 220 Arg Gln Tyr Gln Thr Asp LysLys Glu Gly Thr Asn Asp Ile Val Ala 225 230 235 240 Ser Leu Ser Lys PheGly Arg Asp Phe Val Gly Ser Ile Glu Asn Ala 245 250 255 Ala Gln Thr AspSer Ile Ile Ser Asp Gly Leu Ala Glu Gly Val Gly 260 265 270 Ser Leu LeuGly Ala Gly Pro Val Leu Arg Gly Ala Ser Leu Leu Gly 275 280 285 Lys AlaVal Val Pro Ala Asn Thr Leu Arg Ser Ala Ala Leu Ala Gly 290 295 300 AlaIle Asp Ala Gly Thr Gly Thr Gln Ser Leu Ala Arg Ile Ala Ser 305 310 315320 Thr Val Gly Arg Ala Ala Pro Gly Met Val Gly Val Gly Ala Met Glu 325330 335 Ala Gly Gly Ala Tyr Gln Gln Thr Ala Asp Glu Ile Met Lys Met Ser340 345 350 Leu Lys Asp Leu Glu Lys Ser Pro Val Tyr Gln Gln His Ile LysAsp 355 360 365 Gly Met Ser Pro Glu Gln Ala Arg Arg Gln Thr Ala Ser GluThr Gly 370 375 380 Leu Thr Ala Ala Ala Ile Gln Leu Pro Ile Ala Ala AlaThr Gly Pro 385 390 395 400 Leu Val Ser Arg Phe Glu Met Ala Pro Phe ArgAla Gly Ser Leu Gly 405 410 415 Ala Val Gly Met Asn Leu Ala Arg Glu ThrVal Glu Glu Gly Val Gln 420 425 430 Gly Ala Thr Gly Gln Leu Ala Gln AsnIle Ala Gln Gln Gln Asn Ile 435 440 445 Asp Lys Asn Gln Asp Leu Leu LysGly Val Gly Thr Gln Ala Gly Leu 450 455 460 Gly Ala Leu Tyr Gly Phe GlySer Ala Gly Val Val Gln Ala Pro Ala 465 470 475 480 Gly Ala Ala Arg LeuAla Gly Ala Ala Thr Ala Pro Val Leu Arg Thr 485 490 495 Thr Met Ala GlyVal Lys Ala Ala Gly Ser Val Ala Gly Lys Val Val 500 505 510 Ser Pro IleLys Asn Thr Leu Val Ala Arg Gly Glu Arg Val Met Lys 515 520 525 Gln AsnGlu Glu Ala Ser Pro Val Ala Asp Asp Tyr Val Ala Gln Ala 530 535 540 AlaGln Glu Ala Met Ala Gln Ala Pro Glu Ala Glu Val Thr Ile Arg 545 550 555560 Asp Ala Val Glu Ala Thr Asp Ala Thr Pro Glu Gln Lys Val Ala Ala 565570 575 His Gln Tyr Val Ser Asp Leu Met Asn Ala Thr Arg Phe Asn Pro Glu580 585 590 Asn Tyr Gln Glu Ala Pro Glu His Ile Arg Asn Ala Val Ala GlySer 595 600 605 Thr Asp Gln Val Gln Val Ile Gln Lys Leu Ala Asp Leu ValAsn Thr 610 615 620 Leu Asp Glu Ser Asn Pro Gln Ala Leu Met Glu Ala AlaSer Tyr Met 625 630 635 640 Tyr Asp Ala Val Ser Glu Phe Glu Gln Phe IleAsn Arg Asp Pro Ala 645 650 655 Ala Leu Asp Ser Ile Pro Lys Asp Ser ProAla Ile Glu Leu Leu Asn 660 665 670 Arg Tyr Thr Asn Leu Thr Ala Asn IleGln Asn Thr Pro Lys Val Ile 675 680 685 Gly Ala Leu Asn Val Ile Asn ArgMet Ile Asn Glu Ser Ala Gln Asn 690 695 700 Gly Ser Leu Asn Val Thr GluGlu Ser Ser Pro Gln Glu Met Gln Asn 705 710 715 720 Val Ala Leu Ala AlaGlu Val Ala Pro Glu Lys Leu Asn Pro Glu Ser 725 730 735 Val Asn Val ValLeu Lys His Ala Ala Asp Gly Arg Ile Lys Leu Asn 740 745 750 Asn Arg GlnIle Ala Ala Leu Gln Asn Ala Ala Ala Ile Leu Lys Gly 755 760 765 Ala ArgGlu Tyr Asp Ala Glu Ala Ala Arg Leu Gly Leu Arg Pro Gln 770 775 780 AspIle Val Ser Lys Gln Ile Lys Thr Asp Glu Ser Arg Thr Gln Glu 785 790 795800 Gly Gln Tyr Ser Ala Leu Gln His Ala Asn Arg Ile Arg Ser Ala Tyr 805810 815 Asn Ser Gly Asn Phe Glu Leu Ala Ser Ala Tyr Leu Asn Asp Phe Met820 825 830 Gln Phe Ala Gln His Met Gln Asn Lys Val Gly Ala Leu Asn GluHis 835 840 845 Leu Val Thr Gly Asn Ala Asp Lys Asn Lys Ser Val His TyrGln Ala 850 855 860 Leu Thr Ala Asp Arg Glu Trp Val Arg Ser Arg Thr GlyLeu Gly Val 865 870 875 880 Asn Pro Tyr Asp Thr Lys Ser Val Lys Phe AlaGln Gln Val Ala Leu 885 890 895 Glu Ala Lys Thr Val Ala Asp Ile Ala AsnAla Leu Ala Ser Ala Tyr 900 905 910 Pro Glu Leu Lys Val Ser His Ile LysVal Thr Pro Leu Asp Ser Arg 915 920 925 Leu Asn Ala Pro Ala Ala Glu ValVal Lys Ala Phe Arg Gln Gly Asn 930 935 940 Arg Asp Val Ala Ser Ser GlnPro Lys Ala Asp Ser Val Asn Gln Val 945 950 955 960 Lys Glu Thr Pro ValThr Lys Gln Glu Pro Val Thr Ser Thr Val Gln 965 970 975 Thr Lys Thr ProVal Ser Glu Ser Val Lys Thr Glu Pro Thr Thr Lys 980 985 990 Glu Ser SerPro Gln Ala Ile Lys Glu Pro Val Asn Gln Ser Glu Lys 995 1000 1005 GlnAsp Val Asn Leu Thr Asn Glu Asp Asn Ile Lys Gln Pro Thr Glu 1010 10151020 Ser Val Lys Glu Thr Glu Thr Ser Thr Lys Glu Ser Thr Val Thr Glu1025 1030 1035 1040 Glu Leu Lys Glu Gly Ile Asp Ala Val Tyr Pro Ser LeuVal Gly Thr 1045 1050 1055 Ala Asp Ser Lys Ala Glu Gly Ile Lys Asn TyrPhe Lys Leu Ser Phe 1060 1065 1070 Thr Leu Pro Glu Glu Gln Lys Ser ArgThr Val Gly Ser Glu Ala Pro 1075 1080 1085 Leu Lys Asp Val Ala Gln AlaLeu Ser Ser Arg Ala Arg Tyr Glu Leu 1090 1095 1100 Phe Thr Glu Lys GluThr Ala Asn Pro Ala Phe Asn Gly Glu Val Ile 1105 1110 1115 1120 Lys ArgTyr Lys Glu Leu Met Glu His Gly Glu Gly Ile Ala Asp Ile 1125 1130 1135Leu Arg Ser Arg Leu Ala Lys Phe Leu Asn Thr Lys Asp Val Gly Lys 11401145 1150 Arg Phe Ala Gln Gly Thr Glu Ala Asn Arg Trp Val Gly Gly LysLeu 1155 1160 1165 Leu Asn Ile Val Glu Gln Asp Gly Asp Thr Phe Lys TyrAsn Glu Gln 1170 1175 1180 Leu Leu Gln Thr Ala Val Leu Ala Gly Leu GlnTrp Arg Leu Thr Ala 1185 1190 1195 1200 Thr Ser Asn Thr Ala Ile Lys AspAla Lys Asp Val Ala Ala Ile Thr 1205 1210 1215 Gly Ile Asp Gln Ala LeuLeu Pro Glu Gly Leu Val Glu Gln Phe Asp 1220 1225 1230 Thr Gly Met ThrLeu Thr Glu Ala Val Ser Ser Leu Ala Gln Lys Ile 1235 1240 1245 Glu SerTyr Trp Gly Leu Ser Arg Asn Pro Asn Ala Pro Leu Gly Tyr 1250 1255 1260Thr Lys Gly Ile Pro Thr Ala Met Ala Ala Glu Ile Leu Ala Ala Phe 12651270 1275 1280 Val Glu Ser Thr Asp Val Val Glu Asn Ile Val Asp Met SerGlu Ile 1285 1290 1295 Asp Pro Asp Asn Lys Lys Thr Ile Gly Leu Tyr ThrIle Thr Glu Leu 1300 1305 1310 Asp Ser Phe Asp Pro Ile Asn Ser Phe ProThr Ala Ile Glu Glu Ala 1315 1320 1325 Val Leu Val Asn Pro Thr Glu LysMet Phe Phe Gly Asp Asp Ile Pro 1330 1335 1340 Pro Val Ala Asn Thr GlnLeu Arg Asn Pro Ala Val Arg Asn Thr Pro 1345 1350 1355 1360 Glu Gln LysAla Ala Leu Lys Ala Glu Gln Ala Thr Glu Phe Tyr Val 1365 1370 1375 HisThr Pro Met Val Gln Phe Tyr Glu Thr Leu Gly Lys Asp Arg Ile 1380 13851390 Leu Glu Leu Met Gly Ala Gly Thr Leu Asn Lys Glu Leu Leu Asn Asp1395 1400 1405 Asn His Ala Lys Ser Leu Glu Gly Lys Asn Arg Ser Val GluAsp Ser 1410 1415 1420 Tyr Asn Gln Leu Phe Ser Val Ile Glu Gln Val ArgAla Gln Ser Glu 1425 1430 1435 1440 Asp Ile Ser Thr Val Pro Ile His TyrAla Tyr Asn Met Thr Arg Val 1445 1450 1455 Gly Arg Met Gln Met Leu GlyLys Tyr Asn Pro Gln Ser Ala Lys Leu 1460 1465 1470 Val Arg Glu Ala IleLeu Pro Thr Lys Ala Thr Leu Asp Leu Ser Asn 1475 1480 1485 Gln Asn AsnGlu Asp Phe Ser Ala Phe Gln Leu Gly Leu Ala Gln Ala 1490 1495 1500 LeuAsp Ile Lys Val His Thr Met Thr Arg Glu Val Met Ser Asp Glu 1505 15101515 1520 Leu Thr Lys Leu Leu Glu Gly Asn Leu Lys Pro Ala Ile Asp MetMet 1525 1530 1535 Val Glu Phe Asn Thr Thr Gly Ser Leu Pro Glu Asn AlaVal Asp Val 1540 1545 1550 Leu Asn Thr Ala Leu Gly Asp Arg Lys Ser PheVal Ala Leu Met Ala 1555 1560 1565 Leu Met Glu Tyr Ser Arg Tyr Leu ValAla Glu Asp Lys Ser Ala Phe 1570 1575 1580 Val Thr Pro Leu Tyr Val GluAla Asp Gly Val Thr Asn Gly Pro Ile 1585 1590 1595 1600 Asn Ala Met MetLeu Met Thr Gly Gly Leu Phe Thr Pro Asp Trp Ile 1605 1610 1615 Arg AsnIle Ala Lys Gly Gly Leu Phe Ile Gly Ser Pro Asn Lys Thr 1620 1625 1630Met Asn Glu His Arg Ser Thr Ala Asp Asn Asn Asp Leu Tyr Gln Ala 16351640 1645 Ser Thr Asn Ala Leu Met Glu Ser Leu Gly Lys Leu Arg Ser AsnTyr 1650 1655 1660 Ala Ser Asn Met Pro Ile Gln Ser Gln Ile Asp Ser LeuLeu Ser Leu 1665 1670 1675 1680 Met Asp Leu Phe Leu Pro Asp Ile Asn LeuGly Glu Asn Gly Ala Leu 1685 1690 1695 Glu Leu Lys Arg Gly Ile Ala LysAsn Pro Leu Thr Ile Thr Ile Tyr 1700 1705 1710 Gly Ser Gly Ala Arg GlyIle Ala Gly Lys Leu Val Ser Ser Val Thr 1715 1720 1725 Asp Ala Ile TyrGlu Arg Met Ser Asp Val Leu Lys Ala Arg Ala Lys 1730 1735 1740 Asp ProAsn Ile Ser Ala Ala Met Ala Met Phe Gly Lys Gln Ala Ala 1745 1750 17551760 Ser Glu Ala His Ala Glu Glu Leu Leu Ala Arg Phe Leu Lys Asp Met1765 1770 1775 Glu Thr Leu Thr Ser Thr Val Pro Val Lys Arg Lys Gly ValLeu Glu 1780 1785 1790 Leu Gln Ser Thr Gly Thr Gly Ala Lys Gly Lys IleAsn Pro Lys Thr 1795 1800 1805 Tyr Thr Ile Lys Gly Glu Gln Leu Lys AlaLeu Gln Glu Asn Met Leu 1810 1815 1820 His Phe Phe Val Glu Pro Leu ArgAsn Gly Ile Thr Gln Thr Val Gly 1825 1830 1835 1840 Glu Ser Leu Val TyrSer Thr Glu Gln Leu Gln Lys Ala Thr Gln Ile 1845 1850 1855 Gln Ser ValVal Leu Glu Asp Met Phe Lys Gln Arg Val Gln Glu Lys 1860 1865 1870 LeuAla Glu Lys Ala Lys Asp Pro Thr Trp Lys Lys Gly Asp Phe Leu 1875 18801885 Thr Gln Lys Glu Leu Asn Asp Ile Gln Ala Ser Leu Asn Asn Leu Ala1890 1895 1900 Pro Met Ile Glu Thr Gly Ser Gln Thr Phe Tyr Ile Ala GlySer Glu 1905 1910 1915 1920 Asn Ala Glu Val Ala Asn Gln Val Leu Ala ThrAsn Leu Asp Asp Arg 1925 1930 1935 Met Arg Val Pro Met Ser Ile Tyr AlaPro Ala Gln Ala Gly Val Ala 1940 1945 1950 Gly Ile Pro Phe Met Thr IleGly Thr Gly Asp Gly Met Met Met Gln 1955 1960 1965 Thr Leu Ser Thr MetLys Gly Ala Pro Lys Asn Thr Leu Lys Ile Phe 1970 1975 1980 Asp Gly MetAsn Ile Gly Leu Asn Asp Ile Thr Asp Ala Ser Arg Lys 1985 1990 1995 2000Ala Asn Glu Ala Val Tyr Thr Ser Trp Gln Gly Asn Pro Ile Lys Asn 20052010 2015 Val Tyr Glu Ser Tyr Ala Lys Phe Met Lys Asn Val Asp Phe SerLys 2020 2025 2030 Leu Ser Pro Glu Ala Leu Glu Ala Ile Gly Lys Ser AlaLeu Glu Tyr 2035 2040 2045 Asp Gln Arg Glu Asn Ala Thr Val Asp Asp IleAla Asn Ala Ala Ser 2050 2055 2060 Leu Ile Glu Arg Asn Leu Arg Asn IleAla Leu Gly Val Asp Ile Arg 2065 2070 2075 2080 His Lys Val Leu Asp LysVal Asn Leu Ser Ile Asp Gln Met Ala Ala 2085 2090 2095 Val Gly Ala ProTyr Gln Asn Asn Gly Lys Ile Asp Leu Ser Asn Met 2100 2105 2110 Thr ProGlu Gln Gln Ala Asp Glu Leu Asn Lys Leu Phe Arg Glu Glu 2115 2120 2125Leu Glu Ala Arg Lys Gln Lys Val Ala Lys Ala Arg Ala Glu Val Lys 21302135 2140 Glu Glu Thr Val Ser Glu Lys Glu Pro Val Asn Pro Asp Phe GlyMet 2145 2150 2155 2160 Val Gly Arg Glu His Lys Ala Ser Gly Val Arg IleLeu Ser Ala Thr 2165 2170 2175 Ala Ile Arg Asn Leu Ala Lys Ile Ser AsnLeu Pro Ser Thr Gln Ala 2180 2185 2190 Ala Thr Leu Ala Glu Ile Gln LysSer Leu Ala Ala Lys Asp Tyr Lys 2195 2200 2205 Ile Ile Tyr Gly Thr ProThr Gln Val Ala Glu Tyr Ala Arg Gln Lys 2210 2215 2220 Asn Val Thr GluLeu Thr Ser Gln Glu Met Glu Glu Ala Gln Ala Gly 2225 2230 2235 2240 AsnIle Tyr Gly Trp Thr Asn Phe Asp Asp Lys Thr Ile Tyr Leu Val 2245 22502255 Ser Pro Ser Met Glu Thr Leu Ile His Glu Leu Val His Ala Ser Thr2260 2265 2270 Phe Glu Glu Val Tyr Ser Phe Tyr Gln Gly Asn Glu Val SerPro Thr 2275 2280 2285 Ser Lys Gln Ala Ile Glu Asn Leu Glu Gly Leu MetGlu Gln Phe Arg 2290 2295 2300 Ser Leu Asp Ile Ser Lys Asp Ser Pro GluMet Arg Glu Ala Tyr Ala 2305 2310 2315 2320 Asp Ala Ile Ala Thr Ile GluGly His Leu Ser Asn Gly Phe Val Asp 2325 2330 2335 Pro Ala Ile Ser LysAla Ala Ala Leu Asn Glu Phe Met Ala Trp Gly 2340 2345 2350 Leu Ala AsnArg Ala Leu Ala Ala Lys Gln Lys Arg Thr Ser Ser Leu 2355 2360 2365 ValGln Met Val Lys Asp Val Tyr Gln Ala Ile Lys Lys Leu Ile Trp 2370 23752380 Gly Arg Lys Gln Ala Pro Ala Leu Gly Glu Asp Met Phe Ser Asn Leu2385 2390 2395 2400 Leu Phe Asn Ser Ala Ile Leu Met Arg Ser Gln Pro ThrThr Gln Ala 2405 2410 2415 Val Ala Lys Asp Gly Thr Leu Phe His Ser LysAla Tyr Gly Asn Asn 2420 2425 2430 Glu Arg Leu Ser Gln Leu Asn Gln ThrPhe Asp Lys Leu Val Thr Asp 2435 2440 2445 Tyr Leu Arg Thr Asp Pro ValThr Glu Val Glu Arg Arg Gly Asn Val 2450 2455 2460 Ala Asn Ala Leu MetSer Ala Thr Arg Leu Val Arg Asp Val Gln Ser 2465 2470 2475 2480 His GlyPhe Asn Met Thr Ala Gln Glu Gln Ser Val Phe Gln Met Val 2485 2490 2495Thr Ala Ala Leu Ala Thr Glu Ala Ala Ile Asp Pro His Ala Met Ala 25002505 2510 Arg Ala Gln Glu Leu Tyr Thr His Val Met Lys His Leu Thr ValGlu 2515 2520 2525 His Phe Met Ala Asp Pro Asp Ser Thr Asn Pro Ala AspArg Tyr Tyr 2530 2535 2540 Ala Gln Gln Lys Tyr Asp Thr Ile Ser Gly AlaAsn Leu Val Glu Val 2545 2550 2555 2560 Asp Ala Lys Gly Arg Thr Ser LeuLeu Pro Thr Phe Leu Gly Leu Ala 2565 2570 2575 Met Val Asn Glu Glu LeuArg Ser Ile Ile Lys Glu Met Pro Val Pro 2580 2585 2590 Lys Ala Asp LysLys Leu Gly Asn Asp Ile Asp Thr Leu Leu Thr Asn 2595 2600 2605 Ala GlyThr Gln Val Met Glu Ser Leu Asn Arg Arg Met Ala Gly Asp 2610 2615 2620Gln Lys Ala Thr Asn Val Gln Asp Ser Ile Asp Ala Leu Ser Glu Thr 26252630 2635 2640 Ile Met Ala Ala Ala Leu Lys Arg Glu Ser Phe Tyr Asp AlaVal Ala 2645 2650 2655 Thr Pro Thr Gly Asn Phe Ile Asp Arg Ala Asn GlnTyr Val Thr Asp 2660 2665 2670 Ser Ile Glu Arg Leu Ser Glu Thr Val IleGlu Lys Ala Asp Lys Val 2675 2680 2685 Ile Ala Asn Pro Ser Asn Ile AlaAla Lys Gly Val Ala His Leu Ala 2690 2695 2700 Lys Leu Thr Ala Ala IleAla Ser Glu Lys Gln Gly Glu Ile Val Ala 2705 2710 2715 2720 Gln Gly ValMet Thr Ala Met Asn Gln Gly Lys Val Trp Gln Pro Phe 2725 2730 2735 HisAsp Leu Val Asn Asp Ile Val Gly Arg Thr Lys Thr Asn Ala Asn 2740 27452750 Val Tyr Asp Leu Ile Lys Leu Val Lys Ser Gln Ile Ser Gln Asp Arg2755 2760 2765 Gln Gln Phe Arg Glu His Leu Pro Thr Val Ile Ala Gly LysPhe Ser 2770 2775 2780 Arg Lys Leu Thr Asp Thr Glu Trp Ser Ala Met HisThr Gly Leu Gly 2785 2790 2795 2800 Lys Thr Asp Leu Ala Val Leu Arg GluThr Met Ser Met Ala Glu Ile 2805 2810 2815 Arg Asp Leu Leu Ser Ser SerLys Lys Val Lys Asp Glu Ile Ser Thr 2820 2825 2830 Leu Glu Lys Glu IleGln Asn Gln Ala Gly Arg Asn Trp Asn Leu Val 2835 2840 2845 Gln Lys LysSer Lys Gln Leu Ala Gln Tyr Met Ile Met Gly Glu Val 2850 2855 2860 GlyAsn Asn Leu Leu Arg Asn Ala His Ala Ile Ser Arg Leu Leu Gly 2865 28702875 2880 Glu Arg Ile Thr Asn Gly Pro Val Ala Asp Val Ala Ala Ile AspLys 2885 2890 2895 Leu Ile Thr Leu Tyr Ser Leu Glu Leu Met Asn Lys SerAsp Arg Asp 2900 2905 2910 Leu Leu Ser Glu Leu Ala Gln Ser Glu Val GluGly Met Glu Phe Ser 2915 2920 2925 Ile Ala Tyr Met Val Gly Gln Arg ThrGlu Glu Met Arg Lys Ala Lys 2930 2935 2940 Gly Asp Asn Arg Thr Leu LeuAsn His Phe Lys Gly Tyr Ile Pro Val 2945 2950 2955 2960 Glu Asn Gln GlnGly Val Asn Leu Ile Ile Ala Asp Asp Lys Glu Phe 2965 2970 2975 Ala LysLeu Asn Ser Gln Ser Phe Thr Arg Ile Gly Thr Tyr Gln Gly 2980 2985 2990Ser Thr Gly Phe Arg Thr Gly Ser Lys Gly Tyr Tyr Phe Ser Pro Val 29953000 3005 Ala Ala Arg Ala Pro Tyr Ser Gln Gly Ile Leu Gln Asn Val ArgAsn 3010 3015 3020 Thr Ala Gly Gly Val Asp Ile Gly Thr Gly Phe Thr LeuGly Thr Met 3025 3030 3035 3040 Val Ala Gly Arg Ile Thr Asp Lys Pro ThrVal Glu Arg Ile Thr Lys 3045 3050 3055 Ala Leu Ala Lys Gly Glu Arg GlyArg Glu Pro Leu Met Pro Ile Tyr 3060 3065 3070 Asn Ser Lys Gly Gln ValVal Ala Tyr Glu Gln Ser Val Asp Pro Asn 3075 3080 3085 Met Leu Lys HisLeu Asn Gln Asp Asn His Phe Ala Lys Met Val Gly 3090 3095 3100 Val TrpArg Gly Arg Gln Val Glu Glu Ala Lys Ala Gln Arg Phe Asn 3105 3110 31153120 Asp Ile Leu Ile Glu Gln Leu His Ala Met Tyr Glu Lys Asp Ile Lys3125 3130 3135 Asp Ser Ser Ala Asn Lys Ser Gln Tyr Val Asn Leu Leu GlyLys Ile 3140 3145 3150 Asp Asp Pro Val Leu Ala Asp Ala Ile Asn Leu MetAsn Ile Glu Thr 3155 3160 3165 Arg His Lys Ala Glu Glu Leu Phe Gly LysAsp Glu Leu Trp Val Arg 3170 3175 3180 Arg Asp Met Leu Asn Asp Ala LeuGly Tyr Arg Ala Ala Ser Ile Gly 3185 3190 3195 3200 Asp Val Trp Thr GlyAsn Ser Arg Trp Ser Pro Ser Thr Leu Asp Thr 3205 3210 3215 Val Lys LysMet Phe Leu Gly Ala Phe Gly Asn Lys Ala Tyr His Val 3220 3225 3230 ValMet Asn Ala Glu Asn Thr Ile Gln Asn Leu Val Lys Asp Ala Lys 3235 32403245 Thr Val Ile Val Val Lys Ser Val Val Val Pro Ala Val Asn Phe Leu3250 3255 3260 Ala Asn Ile Tyr Gln Met Ile Gly Arg Gly Val Pro Val LysAsp Ile 3265 3270 3275 3280 Ala Val Asn Ile Pro Arg Lys Thr Ser Glu IleAsn Gln Tyr Ile Lys 3285 3290 3295 Ser Arg Leu Arg Gln Ile Asp Ala GluAla Glu Leu Arg Ala Ala Glu 3300 3305 3310 Gly Asn Pro Asn Leu Val ArgLys Leu Lys Thr Glu Ile Gln Ser Ile 3315 3320 3325 Thr Asp Ser His ArgArg Met Ser Ile Trp Pro Leu Ile Glu Ala Gly 3330 3335 3340 Glu Phe SerSer Ile Ala Asp Ala Gly Ile Ser Arg Asp Asp Leu Leu 3345 3350 3355 3360Val Ala Glu Gly Lys Ile His Glu Tyr Met Glu Lys Leu Ala Asn Lys 33653370 3375 Leu Pro Glu Lys Val Arg Asn Ala Gly Arg Tyr Ala Leu Ile AlaLys 3380 3385 3390 Asp Thr Ala Leu Phe Gln Gly Ile Gln Lys Thr Val GluTyr Ser Asp 3395 3400 3405 Phe Ile Ala Lys Ala Ile Ile Tyr Asp Asp LeuVal Lys Arg Lys Lys 3410 3415 3420 Lys Ser Ser Ser Glu Ala Leu Gly GlnVal Thr Glu Glu Phe Ile Asn 3425 3430 3435 3440 Tyr Asp Arg Leu Pro GlyArg Phe Arg Gly Tyr Met Glu Ser Met Gly 3445 3450 3455 Leu Met Trp PheTyr Asn Phe Lys Ile Arg Ser Ile Lys Val Ala Met 3460 3465 3470 Ser MetIle Arg Asn Asn Pro Val His Ser Leu Ile Ala Thr Val Val 3475 3480 3485Pro Ala Pro Thr Met Phe Gly Asn Val Gly Leu Pro Ile Gln Asp Asn 34903495 3500 Met Leu Thr Met Leu Ala Glu Gly Arg Leu Asp Tyr Ser Leu GlyPhe 3505 3510 3515 3520 Gly Gln Gly Leu Arg Ala Pro Thr Leu Asn Pro TrpPhe Asn Leu Thr 3525 3530 3535 His 16 32 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 16 ggcattacttcatccaaaag aagcggagct tc 32 17 37 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 17 ggccatccat tacttcatcc aaaagaagcggagcttc 37 18 23 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 18 ggatccaaaa gaagcggagc ttc 23 19 32 DNAArtificial Sequence Description of Artificial Sequence Synthetic Primer19 ggcattactt catccaaaag aagctgagct tc 32 20 29 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 20 ggcattacttcatccaaaag aagcggagc 29 21 24 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 21 ggaggctcct cggagtctcc tttt 24 2225 DNA Artificial Sequence Description of Artificial Sequence SyntheticPrimer 22 ggactacctt cgggtagtcc ttttt 25 23 33 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 23 agaagggggctactaagccc tcttcttatt ttt 33 24 19 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Primer 24 aagctgctcc gcagctttt 19 25 35DNA Artificial Sequence Description of Artificial Sequence SyntheticPrimer 25 aaggctatcc ctacgggggt agcctttatt ttttt 35 26 22 DNA ArtificialSequence Description of Artificial Sequence Synthetic Primer 26gccctccttg tgagggcttt tt 22 27 20 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 27 caacgaagcg ttgaatacct 20 28 22DNA Artificial Sequence Description of Artificial Sequence SyntheticPrimer 28 ttcttcgagg cgaagaaaac ct 22 29 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 29 cgacgaggcgtcgaaaacca 20

We claim: 1) A method for detecting a target nucleic acid sequence, themethod comprising: a) providing one or more target probes comprising alinear single-stranded DNA molecule, the target probes comprising atleast two target-complementary sequences that are not joined to eachother, wherein the 5′-end of a first target-complementary sequence iscomplementary to the 5′-end of the target nucleic acid sequence, andwherein the 3′-end of a second target-complementary sequence iscomplementary to the 3′-end of the target nucleic acid sequence, andwherein the target probe that comprises the first target-complementarysequence also comprises a promoter that is joined to the 3′- of thefirst target-complementary sequence, wherein the promoter binds an RNApolymerase that lacks helicase-like activity and that can transcribe RNAusing a single-stranded promoter; b) contacting the target probes withthe target nucleic acid sequence and incubating under hybridizationconditions, such that the target-complementary sequences annealadjacently to the target nucleic acid sequence to form a targetprobe-target complex; c) contacting the target probe-target complex witha ligase under ligation conditions to form a transcription substrate; d)contacting the transcription substrate with the RNA polymerase; e)optionally, repeating steps (a) through (e); and f) detecting thetranscription product. 2) The method of claim 1, wherein the targetnucleic acid sequence comprises a single-stranded DNA molecule obtainedby reverse transcription of RNA. 3) The method of claim 1, wherein thetarget nucleic acid sequence comprises a DNA target nucleic acid in asample. 4) The method of claim 1, wherein the method is used fordetecting an analyte in a sample, wherein the target nucleic acidsequence comprises a target sequence tag that is joined to ananalyte-binding substance, the method further comprising prior to step(a) contacting the analyte-binding substance with the analyte to form aspecific binding pair and separating the specific binding pair fromanalyte-binding substance molecules that are not bound to the analyte.5) The method of claim 4, wherein the analyte is selected from the groupconsisting of a biochemical molecule, a biopolymer, a protein, aglycoprotein, a lipoprotein, an enzyme, a hormone, a biochemicalmetabolite, a receptor, an antigen, an antibody, a nucleic acid, a DNAmolecule, an RNA molecule, a polysaccharide and a lipid. 6) The methodof claim 4, wherein the analyte-binding substance is selected from thegroup consisting of a nucleic acid, a polynucleotide, anoligonucleotide, a segment of a nucleic acid or polynucleotide, a DNAmolecule, an RNA molecule, a molecule comprising both DNA and RNAmononucleotides, modified DNA mononucleotides, a molecule obtained by amethod termed “SELEX”, a nucleic acid molecule having an affinity forprotein molecules, a polynucleotide molecule having an affinity forprotein molecules, an operator, a promoter, an origin of replication, aribosomal nucleic acid sequence, a sequence recognized by steroidhormone-receptor complexes, a peptide nucleic acid (PNA), a nucleic acidand a PNA, a molecule prepared by using a combinatorial library ofrandomized peptide nucleic acids, an oligonucleotide or polynucleotidewith a modified backbone that is not an amino acid, a lectin, a receptorfor a hormone, a hormone, and an enzyme inhibitor. 7) The method ofclaim 1, wherein the target nucleic acid sequence comprises a DNA targetnucleic acid that is a product of an amplification reaction. 8) Themethod of claim 1 wherein the target nucleic acid sequence comprises aproduct of rolling circle replication. 9) The method of claim 7, whereinthe amplification reaction is selected from the group consisting of PCR,RT-PCR, NASBA, TMA, 3SR, LCR, LLA, SDA, Multiple DisplacementAmplification, ICAN™, UCAN™, Loop-AMP, SPIA™ and Ribo-SPIA™. 10) Themethod of claim 1, wherein the one or more target probes comprise abipartite target probe. 11) The method of claim 1 wherein the targetprobe comprising the second target-complementary sequence also comprisesa signal sequence 5′- of said target-complementary sequence. 12) Themethod of claim 11 wherein the signal sequence comprises a substrate forQ-beta replicase. 13) The method of claim 11 wherein the signal sequencecomprises a sequence that encodes a detectable protein. 14) The methodof claim 13 wherein the detectable protein is green fluorescent protein.15) The method of claim 11 wherein the signal sequence comprises asequence that is detectable by a probe. 16) The method of claim 11wherein the signal sequence comprises a sequence that is detectable by amolecular beacon. 17) The method of claim 10 wherein the bipartitetarget probe comprises a transcription termination sequence 5′- of thesecond target-complementary sequence. 18) The method of claim 10 whereinthe bipartite target probe comprises two target-complementary sequencesthat can anneal adjacently to the target nucleic acid sequence. 19) Themethod of claim 1, wherein the one or more target probes comprise apromoter target probe comprising the first target-complementary sequenceand a signal target probe comprising the second target-complementarysequence. 20) The method of claim 19 wherein the signal target probecomprises a signal sequence 5′- of the second target-complementarysequence. 21) The method of claim 20 wherein the signal sequencecomprises a substrate for Q-beta replicase. 22) The method of claim 20wherein the signal sequence comprises a sequence that encodes adetectable protein. 23) The method of claim 22 wherein the detectableprotein is green fluorescent protein. 24) The method of claim 20 whereinthe signal sequence comprises a sequence that is detectable by a probe.25) The method of claim 20 wherein the signal sequence comprises asequence that is detectable by a molecular beacon. 26) The method ofclaim 19 wherein the first target-complementary sequence and the secondtarget-complementary sequence can anneal adjacently to the targetnucleic acid sequence. 27) The method of claim 1 wherein one targetprobe is provided. 28) The method of claim 1 wherein two target probesare provided. 29) The method of claim 1 wherein the number of providedtarget probes is selected from the group consisting of 3, 4, 5, 6, 7, 8,9, and
 10. 30) The method of claim 1 wherein the promoter is an N4 vRNAPpromoter set forth in SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:27, SEQ IDNO:28 or SEQ ID NO:29. 31) The method of claim 1 wherein the promoter isa P2 sequence set forth in SEQ ID NO:16 or SEQ ID NO:28. 32) The methodof claim 1 wherein the ligase is Ampligase® Thermostable DNA Ligase. 33)The method of claim 1 wherein the ligase is selected from the groupconsisting of Tfl DNA Ligase, Tsc DNA Ligase, Pfu DNA ligase, T4 DNAligase and Tth DNA ligase. 34) The method of claim 1 wherein the RNApolymerase comprises a region encoding a polypeptide having an aminoacid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8 or SEQ ID NO:15. 35) The method of claim 1 wherein the RNApolymerase comprises a polypeptide encoded by the nucleic acid sequenceof SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:14.36) The method of claim 1 wherein the RNA polymerase comprises an N4min-vRNAP. 37) The method of claim 1 wherein the RNA polymerasecomprises a domain having 1,106 amino acid residues corresponding toamino acid residues 998-2103 of N4 vRNAP. 38) The method of claim 1wherein the transcription product comprises only ribonucleotides. 39)The method of claim 1 wherein the transcription product comprises atleast one pyrimidine 2′-deoxyribonucleotide having a 2′-substituent onthe sugar moiety. 40) The method of claim 1 wherein the transcriptionproduct comprises at least one pyrimidine2′-fluoro-2′-deoxyribonucleotide. 41) The method of claim 1 wherein thetranscription product comprises pyrimidine2′-fluoro-2′-deoxyribonucleotides. 42) The method of claim 1 wherein thetranscription product comprises AMP, GMP, 2′-F-dUMP and 2′-F-dCMP. 43)The method of claim 1 wherein the transcription product comprises atleast one 2′-amino-2′-deoxyribonucleotide. 44) The method of claim 1wherein the transcription product comprises at least one2′-methoxy-2′-deoxyribonucleotide. 45) The method of claim 1 wherein thetranscription product comprises at least one2′-azido-2′-deoxyribonucleotide. 46) The method of claim 1 wherein thetranscription product comprises at least one2′-amino-2′-deoxyribonucleotide. 47) A method for detecting a targetnucleic acid sequence, the method comprising: a) providing a targetsequence amplification probe (TSA probe) comprising a linearsingle-stranded DNA molecule comprising a 5′-end portion and a 3′-endportion that are not joined, wherein the 5′-end portion is complementaryto the 5′-end of the target nucleic acid sequence, and wherein the3′-end portion is complementary to the 3′-end of the target nucleic acidsequence; b) providing a primer that is complementary to the TSA probe;c) providing one or more target probes comprising a linearsingle-stranded DNA molecule, the target probes comprising at least twotarget-complementary sequences that are not joined to each other,wherein the 5′-end of a first target-complementary sequence iscomplementary to the 5′-end of the target nucleic acid sequence, andwherein the 3′-end of a second target-complementary sequence iscomplementary to the 3′-end of the target nucleic acid sequence; andwherein the target probe that comprises the first target-complementarysequence also comprises a promoter that is joined to the 3′- of thefirst target-complementary sequence, wherein the promoter binds an RNApolymerase that lacks helicase-like activity and that can transcribe RNAusing a single-stranded promoter; d) contacting the TSA probe with thetarget nucleic acid sequence and incubating under hybridizationconditions, such that the end portions anneal adjacently to the targetnucleic acid sequence to form a TSA probe-target complex; e) contactingthe TSA probe-target complex with a ligase under ligation conditions,such that a target sequence amplification circle (TSA circle) is formed;f) contacting the TSA circle with the primer and incubating underhybridization conditions to form a TSA circle-primer complex; g)contacting the TSA circle-primer complex with a strand-displacing DNApolymerase under strand-displacing polymerization conditions, such thata rolling circle replication product comprising multiple copies of thetarget nucleic acid sequence is formed; h) contacting the target probeswith the rolling circle replication product and incubating underhybridization conditions, such that the target-complementary sequencesanneal adjacently to the rolling circle replication product to form arolling circle replication product-target complex; i) contacting therolling circle replication product-target complex with a ligase underligation conditions to form a transcription substrate; j) optionally,releasing the transcription substrate from the rolling circlereplication product complex, k) contacting the transcription substratewith the RNA polymerase under transcription conditions to form atranscription product; l) optionally, repeating steps (a) through (k);and m) detecting the transcription product. 48) A method for selectivelytranscribing a target nucleic acid sequence, the method comprising a DNAligation operation and a transcription operation, wherein the DNAligation operation comprises ligation of one or more target probescomprising a promoter that is 3′- of a target complementary sequence,which promoter binds an RNA polymerase that lacks helicase-like activityand that can transcribe RNA using a single-stranded promoter to form atranscription substrate, wherein the ligation is dependent onhybridization of the target probes to the target nucleic acid sequence,to form a transcription substrate and wherein the transcriptionoperation comprises contacting the transcription substrate with the RNApolymerase. 49) A method for detecting a target nucleic acid sequence,the method comprising: a) providing one or more target probes comprisinga linear single-stranded DNA molecule, the target probes comprising atleast two target-complementary sequences that are not joined to eachother, wherein the 5′-end of a first target-complementary sequence iscomplementary to the 5′-end of the target nucleic acid sequence, andwherein the 3′-end of a second target-complementary sequence iscomplementary to the 3′-end of the target nucleic acid sequence, andwherein the target probe that comprises the first target-complementarysequence also comprises a promoter that is joined to the 3′-end of thefirst target-complementary sequence, which promoter binds an RNApolymerase that lacks helicase-like activity and that can transcribe RNAusing a single-stranded promoter; b) contacting the target probes withthe target nucleic acid sequence and incubating under hybridizationconditions, such that the target probes anneal to the target nucleicacid sequence to form a target probe-target complex; c) contacting thetarget probe-target complex with a DNA polymerase under DNApolymerization conditions to form one or more DNA polymerase extensionproducts that are adjacent to the 5′-end of a target-probe, such that acomplex is formed; d) contacting the complex with a ligase underligation conditions to form a transcription substrate; e) contacting thetranscription substrate with the RNA polymerase to form a transcriptionproduct; f) optionally, repeating steps (a) through (f); and g)detecting the transcription product. 50) A kit for detecting a targetnucleic acid sequence, the kit comprising: a) one or more target probescomprising a linear single-stranded DNA molecule, the target probescomprising at least two target-complementary sequences that are notjoined to each other, wherein the 5′-end of a first target-complementarysequence is complementary to the 5′-end of the target nucleic acidsequence, and wherein the 3′-end of a second target-complementarysequence is complementary to the 3′-end of the target nucleic acidsequence, and wherein the target probe that comprises the firsttarget-complementary sequence also comprises a promoter that is joinedto the 3′- of the first target-complementary sequence, wherein thepromoter binds an RNA polymerase that lacks helicase-like activity andthat can transcribe RNA using a single-stranded promoter; b) a ligase;and c) the RNA polymerase. 51) The kit of claim 50 further comprising areverse transcriptase. 52) The kit of claim 50 further comprising atarget sequence amplification probe (TSA probe) comprising a linearsingle-stranded DNA molecule comprising a 5′-end portion and a 3′-endportion that are not joined, wherein the 5′-end portion is complementaryto the 5′-end of the target nucleic acid sequence, and wherein the3′-end portion is complementary to the 3′-end of the target nucleic acidsequence; a primer that is complementary to the target sequenceamplification probe, and a strand-displacing DNA polymerase. 53) The kitof claim 50 further comprising a DNA polymerase.